MSU LIBRARIES .—,‘—. RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wil] be charged if book is returned after the date stamped below. EFFECTS OF EXPOSURE TO POLYBROMINATED BIPHENYLS ON URINARY STEROID METABOLIC PROFILES IN MAN AND RATS AS DETERMINED BY CAPILLARY GC AND GC/MS/DS By John James Vrbanac, Jr. A DISSERTATION Submitted to Michigan State University in partial fquiTIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1983 ABSTRACT EFFECTS OF EXPOSURE TO POLYBROMINATED BIPHENYLS ON URINARY STEROID METABOLIC PROFILES IN MAN AND RATS AS DETERMINED BY CAPILLARY GC AND GC/MS/DS By John James Vrbanac, Jr. Polyhalogenated organic compounds have been shown to be potent inducers of mixed-function oxidases (MFOs) responsi- ble for the metabolism of a wide range of compounds includ- ing steroid hormones. Since induction of MFO enzyme systems increases the metabolism of steroids and since alterations in steroid hormone secretion and metabolism may be reflected by changes in the urinary steroid metabolic profile, the effects of exposure to PBBs (potent MFO inducers) on steroid hormone excretion were investigated by examining urinary steroid metabolic profiles in exposed humans and in an experimental model (rats). Advantages and disadvantages of using capillary column GC/FID and packed and capillary columns GC/MS/MSSMET (an off-line reverse library search analysis) were investigated. Capillary GC/FID was adequate for quantitative analysis of most steroids isolated from human urine. The major drawback to using capillary GC/FID was the time involved in analysis of the GC traces. Generation of human urinary steroid metabolic profiles by GC/MS/MSS was faster, but a major drawback was the relative insensitivity of this method compared to capillary column GC/FID. Exposure of male and female rats to PBBs etc was observed to alter the urinary steroid metabolic profile in the corticosteroid region of the profile. Urinary 63-hydroxycortisol, a well-characterized indicator hepatic cytochrome P-450 mixed function oxidase activity, was increased in the PBBs-exposed humans (non-smoking healthy adult male subjects with greater than 50 ppb P885 in their serum). This effect was less than that seen following chronic exposure to high therapeutic doses of phenobarbital, but was similar in magnitude to increases in urinary 6e-hydroxycortisol excretion reported for workers exposed to DDT. Certain other steroids, all but one of adrenal origin, appeared to be elevated in the P385 exposed population. ACKNOWLEDGEMENTS The author gratefully acknowledges the staff and students associated with the National Institutes of Health/Michigan State University Mass Spectrometry Facility, especially Millie Martin, Linda Seymour, Jennifer Valentine, Dave Pinkston, Lee Westover, Mike McPherson, Rick Chapman, Brian Musselman, Vicki McPharlin, Charles C. Sweeley, J. Throck Watson and John F. Holland. My gratitude is also extended to all committee members, Theodore M. Brody, W. Emmett Braselton, Jr., John F. Holland, J. Throck Watson and Jerry B. Hook, for extending to me their valuable time, and to Karen DeFord for preparation of this manuscript. Special thanks to my major advisor, W. Emmett Braselton, Jr., and to Charles C. Sweeley, for their expert guidance throughout this project. Finally, I extend my sincere gratitude to my wife, Patricia, and to Mary Ellen and J.D. for support and friendship. ii TABLE OF CONTENTS -PRELIMINARY PAGES- Acknowledgements. . . . . . . . . . . . . . . . List of Tables. . . . . . . . . . . . . . . List of Figures . . . . . . . . . . . . . . —TEXT PAGES- SECTION I. General Introduction. . . . . . . . A. Objectives. . . . . . . . . . . . . . B. Background. . . . . . . . . . . . . . 1. Mixed function oxidases . . . . 2. Effects of xenobiotics on steroid metabolism and related subjects . 3. Metabolic profiling . . . . . . C. Rationale . . . . . . . . . . . . . SECTION II. Development of Analytical Methods. A. Introduction. . . . . . . . . . . . . 8. Experimental. . . . . . . . . . . . . C. Results and Discussion. . . . . . SECTION 111. Animal Studies. . . . . . . . . . A. Introduction. . . . . . . . . . . . . B. Experimental. . . . . . . . . . . . C. Results . . . . . . . . . . . . . . . D. Discussion. . . . . . . . . . . . . . iii ii vi I6 37 38 38 40 49 83 83 92 93 121 Table of Contents (continued) SECTION IV. Human Studies. . A. Introduction . . . . B. Experimental. . . . . C. Results . . . . . . . D. Discussion. . . . . . SECTION V. APPENDIX I. APPENDIX 11. Information . APPENDIX III. LITERATURE CITED. . . . . . Glossary of Terms. iv Conclusions and Direction Individual Human Subject Health Michigan Department of Public Health, Division of Environmental Epidemiology Health Questions. 123 123 130 132 162 172 178 186 190 191 LIST OF TABLES TABLE PAGE 1 Precision: GC profiling of urinary steroids. . . . . . . . 52 2 Precision: Capillary column GC/MS profiling of urinary SterOTds ....... O O O O O O O O 0 O O O O ..... 68 3 Effect of chronic exposure to PBBs on female rat urinary SterOids ......... I O O O O O O O O ....... 96 4 Changes in urinary excretion of some corticosterone metabolites in female rats following acute exposure to PBBS. O O O I O O O O 0 O 0 O I O O O O I O O O O O O O 99 5 Urinary metabolites of corticosterone in control and P885 exposed adult male rats . . . . . . ......... 114 6 Urinary steroid metabolic profiles of a normal adult male: 24 hour excretion patterns over a 6-day period . . . 133 7 Urinary steroid metabolic profiles of a normal adult male: Morning spot urine excretion patterns over a 6-day periOd ccccc o o o o o o o o o o o ooooooo 136 8 Average urinary excretion patterns over a 6-day period in absolute amounts for a 24-hour urine collection relative to creatinine and a morning spot urine collection ...... . . . . . . . . . . . . . . . . . . 138 9 Determination of 6@-hydroxycortisol by automated reverse library search of selected mass chromatograms. . . 146 10 Human urinary steroid metabolic profiles of non-smoking male subjects: Average plus standard deviation, normalized to the internal standard and urinary creatinine . . . . . . . . . . . . . . . . . . . ..... 153 11 Unknowns elevated in the urine of P885 exposed human subjects as determined by capillary GC with FID. . . . . . 154 12 Human urinry steroids in controls, PBBs exposed and phenobarbital exposed individuals as determined by GC/MS/DS O O O O O O O O O O I O 0 O O O O O O 0 O I O O O 156 13 Possible consequences of induction and/or inhibition of liver microsomal enzyme systems . . . . . . ...... 166 FIGURE 1 #00 10 11 12 13 14 LIST OF FIGURES PAGE GC/MS/DS analyses of urinary steroids from a normal female ........ . ................. 18 Description of MSSMET library files ........... 22 Description of found file ................ 25 Description of the normalized found file ......... 29 Example of a typical MSSHET quantitative printout (Found File) obtained for a normal female human urinary steroid sample. . . . . . . . . . .......... . ..... 30 Flow diagram for isolation and chemical derivatization of urinary steroids by a modification of the method of Leunissen and Thijssen (1978) .............. 45 Urinary steroid metabolic profile of a post-puberal pre-menopausal female human obtained by capillary column GC with flame ionization detection. . . . . . . . . . . . 50 Urinary steroid metabolic profile of a normal adult male subject obtained using a 60 m DB-l bonded phase fused silica capillary column ......... . . ...... 54 GC/MS analysis of aliphatic hydrocarbon standards and urinary steroids using a 3 m, 3% OV-101 packed column . . 57 GC/MS analysis of urinary steroids in the region of RI 2500-3100 using a 3 m, 3% 0V-101 packed column ...... 60 GC/MS analysis of urinary steroids using a 50 m (0.3 mm 1.0.) OV-101 WCOT fused silica capillary column . . . . . 62 GC/MS analysis of urinary steroids using a 50 m OV-lOl NCOT fused silica capillary column .......... . . 65 Urinary steroid metabolic profile of a normal adult male SUbject 0 O ...... O I O O 0 O O O O O I O O O O O O 70 Changes in peak shape and number of points across a gas chromatogarphic peak following increasing amounts of n-pentadecane injected into a 25 m OV-101 fused silica capillary column (0.32 mm 1.0., 150°C isothermal) . . . . 74 LIST OF FIGURES (continued) 16 17 18 19 20 21 22 23 24 25 26 27 28 Total ion intensity v. scan number in the region of the chromatogram where Ila-hydroxyandrosterone, 118-hydroxy- etiocholanolone and the internal standard elute . . . . . Urinary steroid metabolic profiles using a 25 m OV-lOl fused silica capillary column with FID (top trace) and the same column with MS detection (repetitive scanning; 2 s scan cycle) . . . . . . . . . . . . . . . . . . . . . Urinary steroid metabolic profiles of female rats exposed to 100 ppm PBBs chronically (top trace) and of an appropriate control group. . . . . . . . . . . . . . . Mass spectrum of the MO-TMS derivative of a tetrahydroxy- pregnane-one that increased in the urine of P885 exposed fen'a] 8 rats 0 O I O O O O O O I I O I O O O I O I O O O 0 Mass spectrum of the MO-TMS derivative of a tetrahydroxy- pregnane-one that decreased in the urine of P885 exposed fema1e rats 0 O O O O O O O O O O O O O O O O O O O O O 0 Mass spectrum of the MO-TMS derivative of a pentahydroxy- pregnane-one that decreased in the urine of P885 exposed fema] e rats 0 O O O O O O O O O O O I O O O O O O O I O O Capillary column GC traces obtained for three different fractions from Sephadex LH-20 column chromatography . . . Capillary column GC traces obtained for two different fractions from Sephadex LH-20 column chromatography . . . Mass spectrum of a Sa-pregnane-3,11,16,20,21-pentol . . . Capillary GC trace of urinary steroids obtained for a group of four male rats before (control) and after ("germ-free") antibiotic treatment. . . . . . . . . . . . Electron impact mass spectrum of the methoxime-trimethyl- silyl derivative of 63-hydroxycortisol. . . . . . . . . . Dot plots of urinary concentrations of 6e-hydroxycortisol in phenobarbital and control groups . . . . . . . . . . . Dot plots of urinary concentrations of 65-hydroxycortisol in PBBs exposed and control groups. . . . . . . . . . . . Plots of Student's t-statistic when urinary steroid data are randomly reassigned . . . . . . . . . . . . . . . . . vii PAGE 76 78 94 100 102 104 109 111 116 118 145 148 150 168 SECTION I General Introduction SECTION I OBJECTIVES The long range goal of this work was to develop a non- invasive procedure for the identification of individuals with significant xenobiotic-induced alterations in steroid metabolism. Initial efforts focused on development of capillary column gas chromatography (GC; a glossary of terms is listed in Appendix III) with flame ionization detection (F10) and gas chromatography/mass spectrometry/data system (GC/MS/DS) methods for qualitative and quantitative analysis of urinary steroids. It was believed that these methods would allow certain xenobiotic-induced changes in steroid hormone metabolism to be monitored using urine samples. It has been demonstrated that many environmental chemicals that either stimulate or inhibit mixed function oxidase (MFO) enzyme systems alter steroid hormone metabolism in a similar manner. For this project polybrominated biphenyls (PBBs) were chosen as a model to represent xenobiotic stimulators of MFO enzyme systems. The immediate objectives of this project were to: 1) DevelOp a quantitative procedure for the isolation, purification and derivatization of steroids from urine and plasma. 2) 3) 4) Develop capillary column GC and an automated GC/MS/DS technique for the quantitative analysis of a mixture of steroids isolated from urine or plasma. Use the rat as a model to examine the effects of P885 on the urinary steroid metabolic profile and correlate these changes with known specific effects these chemicals have on steroid metabolism. Extend the study to a human population by examining the steroid metabolic profiles of a group of subjects accidentally exposed to PBBs. BACKGROUND 1. Mixed function oxidase enzyme systems: The term "mixed-function“ oxidase was first used by Mason (1957) to describe certain types of biological oxidations. This term describes particular enzyme complexes which require molecular oxygen and NADPH and which exhibit a wide range of substrate specificities. In these reactions one atom of oxygen appears in the metabolized substance and one atom of oxygen appears in water. Steroid hormone lyases and hydroxylases are part of this general enzyme system. The mixed-function oxidase sytem is also responsible for the oxidation of lipids and for the oxidative detoxication of a large number of harmful xenobiotics. In general, the mixed- function oxidase enzymes responsible for the oxidations of xenobiotics, lipids and steroids are associated with smooth endoplasmic reticulum, although their synthesis is apparent- ly associated with the rough endoplasmic reticulum. A carbon monoxide binding pigment in liver microsomes was first described by Klingenberg (1958) and Garfinkel (1958). This pigment was later named cytochrome P-450 since it was observed to have an absorption maximum at 450 nm when combined with carbon monoxide and was shown to be a proto- porphyrin hemOprotein of the cytochrome b-5 type (Omura and Sato, 1964). It is now recognized that the cytochrome P-450 system is involved in the transfer of electrons to oxygen to form water and oxidized product in the above mentioned mixed-function oxidase systems, and that there is a wide range of substrates of endogenous and exogenous origin that undergo oxidation, reduction or are otherwise metabolized (i.e., clevage reactions for example) by cytochrome P-450 complexes (Niekramasinghe, 1975; Blumberg, 1978; Makin, 1975; Schulster, 1976). Cytochrome P-450 participation in steroid hydroxylation reactions has been shown to occur in the mitochondria and microsomes of those tissues associated with steroid synthesis (Niekramasinghe, 1975; McIntosh gt 21., 1971 and 1973; Mason ££._l-: 1973; Meigs and Ryan, 1968). The concentration and distribution of the various cytochrome P-450 enzyme complexes is quite dependent upon such variables as sex, age, metabolic state and environ- mental factors (McIntosh gt 31., 1973; McKerns, 1968). It is well established that there are multiple forms of cytochrome P-450 for the metabolism of substances of both endogenous and exogenous origin (wiekramasinghe, 1975; Blumberg, 1978). Sladek and Mannering (1966) were the first to obtain evidence for more than one form when they noticed that changes in the carbon monoxide difference spectrum occurred when animals were pretreated with polycyclic aromatic hydrocarbons. The carbon monoxide difference spectrum maximum had shifted from 450 mu to 448 mu in these experiments and this hemoprotein has come to be termed cytochrome P-448. Two classical types of microsomal mixed- function oxidase enzyme induction have evolved using the above classification. Phenobarbital is the classical inducer of the P-450 type cytochrome and 3-methylchol- anthrene (3-MC) is the classical inducer of the P-448 type cytochrome (Sladek and Mannering, 1969a, 1969b). 2,3,7,8- Tetrachlorodibenzo-p-dioxin (TCDD) is an agent that also belongs to the 3-MC classification of inducer. The 3-MC type induction affects the metabolism of a smaller number of compounds than the phenobarbital type induction. In some cases, pretreatment of subjects with a specific inducer will affect the pharmacological/toxicological response to a second agent (the effect can be either augmented or attenu- ated). For example, barbiturate-induced sleeping time is reduced by pretreatment of subjects with phenobarbital. Phenobarbital-like induction also results in proliferation of the endoplasmic reticulum. This type of induction has been characterized by a number of enzyme assays, aminopyrine-N-demethylase and epoxide hydrolase being two of the better known examples. 3-MC-like induction results in an abnormal liver pathology and has also been characterized using a number of specific enzyme assays (benzoEaprrene hydroxylase and UDP-glucuronyl-p-nitrophenol glucuronyl transferase for example). 2. Effects of xenobiotics on steroid metabolism and related subjects: It is well established that many different drugs and environmental chemicals affect both hepatic and extrahepatic MFO activity (Conney, 1971, 1976; Conney gt gl., 1973). These effects can result in an alteration in the intensity and duration of action of drugs metabolized by the systems. For example, phenobarbital, benzoEaprrene, DDT, poly- chlorinated biphenyls (PCBs), phenylbutazone and aminopyrene stimulate oxidative drug metabolizing enzymes in liver microsomes while chloramphenicol, chlorthion, carbon monoxide, and bis-hydroxycoumarin inhibit these enzymes 1., 1973). Since the microsomal MFO system is (Conney gt important in steroid hormone biochemistry and xenobiotic induction of MFOs has been shown to affect steroid metabolism it follows that xenobiotic-induced alterations in MFO activity could influence endocrine balance to the extent that biological functions such as reproduction may be influenced. Induction by PCBs. Contamination of much of the ecosystem by PCBs is considered to be a serious environ- mental concern. PCBs are lipid soluble compounds and are chemically stable. PCBs have been observed to cross the placental barrier and have been found in milk (Orberg, 1977; Allen and Barsotti, 1976; Kuratsune gt gl., 1972; Takagi gt gl., 1976). PCBs increased the hepatic metabolism of testosterone, 4-androstene-3,17-dione and estradiol-17s in the chicken (Nowicki and Norman, 1972). This altered hepat- ic metabolism of steroids is believed to be the cause of PCBs-induced egg shell thinning in certain species of birds (Haegele and Tucker, 1974) since the plasma concentrations of estrogens and progesterones are believed to be critical in this process. Rats, mice and rhesus monkeys treated with PCBs showed elevated corticosterone concentrations and increased size of the adrenals (Wasserman gt gl., 1973; Sanders gt gl., 1974; Barsotti and Allen, 1975). Interes- tingly, in one study the "total plasma corticoids" in mice was reported to be slightly reduced (Sanders and Kirk- patrick, 1975). Exposure to PCBs resulted in decreased urinary dehydroepiandrosterone in the boar (Plantonow gt gl., 1972). PCBs also increased the catabolism of exogen— ously administered progesterone, estradiol-173 and testos- terone in rodents, an effect attributed to PCBs-induced increases in microsomal enzyme activity (Orberg and Kihlstrom, 1973; Orberg and Lundberg, 1974; Orberg and Ingvast, 1977; Derr, 1978). In general, the highly chlorin- ated PCBs have a greater stimulatory effect on microsomal MFO enzymes than the lowly chlorinated PCBs (Bickers _t gl., 1974; Orberg, 1976). Thus, PCBs affect the metabolism of androgens, estrogens and corticosteroids and would therefore be expected to affect physiological processes regulated by these steroids. Exposure to PCBs has been observed to result in a variety of toxicities to the reproductive system. Women exposed to PCBs and other chlorinated hydrocarbons have shown menstral cycle irregularities and dysmenorrhea (Kuratsune gt gl., 1972) and rhesus monkeys exposed to PCBs showed lengthened menstral cycles (Allen and Barsotti, 1976). Female mice exposed to PCBs showed a reduction in the number of ova implanted following mating and a length- ened estrus cycle (Orberg and Kihlstrom, 1973; Orberg gt _l., 1972) and pregnant rabbits treated with PCBs showed reduced litter sizes (Villeneuve gt gl., 1971a and 1971b). Certain of the lowly chlorinated PCBs show estrogenic activity, an effect which is apparently obscured in complex PCBs mixtures by altered steroid metabolism and other factors (Nelson, 1974; Bitman and Cecil, 1970; Gellert, 1978). Thus, PCBs decrease the reproductive capability of mammals, an effect that at least, in part, probably relates to effects on steroid hormone metabolism. Induction by PBBs. Polybrominated biphenyls (PBBs) were accidentally added to dairy cow feed in Michigan resulting in a contamination of the food supply (Dunckel, 1975). Like PCBs, PBBs accumulate in milk and fat tissues and are chemically stable (Fries and Marrow, 1974; Matthews gt gl., 1977, 1978; Rickert gt gl., 1978). Also, like PCBs, PBBs are potent stimulators of liver and extrahepatic microsomal MFO enzyme systems (Dent gt _l., 1976; McCormack _t g1 1978, 1979; Arneric gt fl” 1980; Newton 3531., 1980). Initial studies investigating PBBs indicated that they had characteristics similar to the TCDD-type toxic polyhalo- genated aromatic-hydrocarbons and also the classical inducer phenobarbital and therefore were classified as "mixed" inducers (Dent, 1976; Sladek and Mannering; 1969a, 1969b). PBBs were noted to be inducers of liver microsomal aryl hydrocarbon hydroxylase as well as enzymes which are induced by phenobarbital. PBBs exposure was observed to elicit a toxicity syndrome similar to TCDD in that PBBs caused 1) pathological changes in the liver similar to those seen following TCDD, 2) depressed body weight gains, 3) were immunotoxic, 4) and produced edema in chicks (Jackson and Halbert, 1974; Ringer and Polin, 1977). Because PBBs are a mixture of various congeners with different chemical proper- ties, it was of interest to define the chemical nature of the mixture and the biological effects of the various PBBs congeners present in Firemaster BP-6 and other commercial PBBs preparations. The particular commercial mixture of P885 which contaminated Michigan contained 10 components each of which made up greater than 1% of the mixture, with three of these components accounting for 75% of the mixture (Moore and Aust, 1978; Moore gt gl., 1978, 1980). These considerations are important since the chemical and toxico- logical properties of individual pure congeners are highly variable. In particular, those congeners with 2 or more ortho bromines are relatively non—toxic and have a phenobar- bital-like effect in that they induce liver microsomal enzymes usually induced by phenobarbital and cause a 10 proliferation of hepatic endoplasmic reticulum. Congeners without bromines in the ortho position have a toxicity similar to T000 and induce microsomal enzymes usually associated with 3-methylcholanthrene exposure. Those congeners with one ortho bromine show moderate toxicity and were "mixed“ inducers of liver microsomal enzymes, having both 3-methylcholanthrene phenobarbital-like effects (Dannan _t _l., 1978; Moore gt gl., 1980). Individual components of the commercial mixture of P885 which contaminated Michigan all contained at least one ortho bromine and 89% of the total mixture contained two ortho bromines. Thus, it would be reasonable to expect that one of the possible effects of this particular mixture of P885 would be a "phenobarbital- like" effect in the exposed human population. PBBs reduced the response to exogenous steroid sex hormones (McCormack gt gl., 1979). PBBs can undergo trans- placental movement and, being lipid soluble, are present in the milk of exposed lactating females. The offspring of rats fed a diet containing PBBs from the 8th day of pregnan- cy until 28 days postpartum had significant concentrations of P885 in liver, kidney and fat tissues 300 days later (McCormack gt gl., 1980a, 1980b). The offspring also showed renal and hepatic enzyme stimulation, a reduction in pento- barbital sleeping time and histopathological alterations. Dietary PBBs administration to rats by the above regime altered ifl liELQ metabolism of progesterone by hepatic microsomal MFOs in the offspring (Arneric gt gl., 1980). 11 In the same study PBBs were reported to increase the metabo- lism of progesterone to both 16a- and 68-hydroxyproges- terone. This effect was sex dependent in immature animals and resembled the MFO stimulatory effects of phenobarbitol more that 3-methylcholanthrene in this system. Metabolism of estradiol, estrone and ethynyl estradiol by hepatic microsomes was also increased by exposure to PBBs (Newton gt I_l., 1980; Bonhaus gt gl., 1982). The effects of P885 treatment on the activities of testosterone 16a-hydroxylase, 7a-hydroxylase, 68-hydroxylase and 173-dehydrogenase were also studied using the above treatment protocol. 7a-Hydroxylase activity was stimulated by P885 in males and females. This effect was more pronounced in females at 2 and 4 months of age. 16a-Hydroxylase activity was increased in females of all ages while only the immature males showed an increase in activity. 68-Hydroxylase activity was increased in both sexes at all ages. 178-Dehydrogenase activity was elevated at all ages in treated females and in one month old males. Interestingly, 10 ppm PBBs increased 16a-hydroxylase and 178-dehydrogenase activities in 8 week old males but 100 ppm PBBs did not, suggesting that high concentrations of P885 may actually inhibit steroid metabo- lism of these two enzymes In the same series of studies (Newton gt gl., 1980, 1982), PBBs also decreased the reduc- tive metabolism of testosterone to the more potent androgens Sa-dihydrotestosterone and Sa—dihydroandrostenedione. 12 As one might expect, the reproductive toxicities associated with PCBs have also been seen following PBBs exposure. PBBs administration lengthened the estrus cycles in rats and rhesus monkeys (Johnston gt 31., 1980; Lambrecht _t _1., 1978). The effect in monkeys was associated with attenuated serum progesterone peaks (Lambrecht gt g1., 1978). Perinatal PBBs exposure delayed vaginal opening in rats (McCormack gt g1., 1980b; Harris gt g1., 1978). PBB exposure reduced spermatogenesis and caused testicular atrophy in bulls, and resulted in hypoactive seminiferous tubules in rhesus monkeys (Allen gt g1., 1978; Jackson and Halbert, 1974). Thus, like PBCs, exposure to PBBs stimulated the metabolism of androgens and estrogens and interfered with reproductive processes. PBBs treatment has been observed to have significant effects on the metabolism of sex steroids. PBBs increased the conversion of progesterone to 160' and 63-hydroxy- progesterone and increased the oxidative metabolism of estradiol, estrone and ethynyl estradiol. PBBs treatment also increased testosterone metabolism and induced a reduc- tion in androgenicity of testosterone, an effect which may be brought about in three ways: 1) decreased Sa-reduction of testosterone to Sa-dihydrotestosterone and Sa-dihydro- androsterone, 2) enhanced oxidation of testosterone by 68-, 7a- and 166-hydroxylases, 3) enhanced 17B-oxidation to androstenedione. 13 Since the major route of elimination of conjugated and unconjugated metabolites of steroid hormones is through renal excretion, the above xenobiotic-induced changes in steroid hormone metabolism should be reflected in the amounts of steroid metabolites in the urine. For example, increased urinary excretion of 16a- and 63-hydroxylated metabolites of progesterone and 16a-, 7a- and 68-hydroxylat- ed metabolites of testosterone might be observed in PBBs- treated subjects. In general, one might expect that large increases in the metabolism of any steroid hormone should be reflected by increased urinary excretion of its metabolites. Many compounds have been reported to inhibit microsomal MFO activity. For example, the halogenated hydrocarbons chlorthion and carbon tetrachloride decreased microsomal MFO activity (Conney gt g1., 1973). Certain heavy metals such as cadmium, lead and organic mercurial compounds also decreased MFO activity (Conney gt g1., 1973; Means and Schnell, 1979, 1980; Meredith gt g1., 1977; Ahotupa _t g1., 1979; Aitio gt g1., 1978). Cadmium treatment caused a marked inhibition of hepatic microsomal MFO drug metabolizing enzyme activity (Means and Schnell, 1980; Hadley gt g1., 1974; Krasny and Holbroot, 1977) and has reduced testicular synthesis of testosterone both 13 tilt and it XIELE (Chandler gt g1,, 1976; Gunn gt g1., 1965). However, perinatal cadmium exposure increased both adrenal steroidogenesis and the hepatic reduction of corticosterone in adult rats, an effect that was suggested to be due to a 14 general increase in the activity of hepatic steroid reduc- t al., 1978). Such an effect is interesting tases (Grady in view of the fact that PBBs inhibited hepatic steroid reductases (McCormack gt g1., 1978; McCormack gt g1., 1979) In contrast to cadmium, chronic administration of methyl mercury resulted in a decreased hepatic metabolism of corticosterone and a decreased synthesis of steroids in the adrenal gland (Grady gt g1., 1978; Burton and Meikle, 1977) Cadmium, methyl mercury and lead were also toxic to both the male and female reproductive system (Lancranjan, 1975; Lucier g_ g1., 1977; Harbison gt g1., 1978). Grady gt g1. (1978) showed that cadmium effects on corticosterone reduc- tion and adrenal steroidogenesis were correlated with increased plasma corticosterone and adrenal weights. Ten ppm cadmium in drinking water was reported to completely prevent reproduction in a colony of mice (Schroeder and Mitchener, 1971). Thus, many different xenobiotics can cause significant changes in both hepatic and extrahepatic enzyme systems that metabolize steroid hormones. It appears likely that although the mechanisms of action of these compounds are indeed very complex, at least part of their toxicities relates to altered metabolism of steroid hormones. This is especially true for some of the polyhalogenated hydro- carbons. In preliminary studies, PBBs treatment affected the profile of steroid metabolites in the urine of rats. These observations lead one to suspect that physiologically 15 significant changes in steroid metabolism caused by exposure to environmental chemicals should result in a significant alteration in the excretion of steroid metabolites and that such changes may be detected by examining urinary steroids. Metabolism of PBBs. The endoplasmic reticulum is the site of metabolism for a large number of drugs and xeno- biotics and is probably where metabolism of certain PBB congeners occurs. 13 liLLE studies indicate that at least two of the major congeners in Firemaster BP-6 can undergo rapid metabolism in rat hepatic microsomal preparations isolated from subjects pretreated with phenobarbital. Microsomal preparations from nontreated and 3-MC pretreated subjects were ineffective (Dannan gt g1., 1978; Dannan and Aust, 1983). These particular congeners are the only two in the Firemaster BP-6 mixture known to have at least one unsubstituted para carbon. In these experiments the reac- tion rate was determined by substrate disappearance (measured by GC) and the chemical nature of the metab- olite(s) was not determined (aromatic hydroxylation would be the most likely reaction to be occurring). Metabolism of some PBB congeners not found in the Firemaster BP-6 mixture has been investigated (Safe gt g1., 1976; Kohli and Safe, 1976; Kohli gt g1., 1978) but will not be discussed here. 16 3. Metabolic Profiling: General Concepts. The concept that individuals have distinct metabolic patterns reflected by the constitutents of their biological fluids (i.e., urine, blood, amniotic fluid, cerebrospinal fluid, sweat, etc.) originated with the work of Roger Williams (1951). By utilizing the technique of paper chromatography, Nilliams showed convincingly that the patterns for a variety of compounds found in urine varied greatly from one individual to another but were rela- tively constant for any given individual. William's concept was not employed by others until the late 1960's when liquid and gas chromatographic techniques had become refined enough to allow for studies of this type with considerably less effort. The phrase most often used to describe multi- component analysis of biological fluids, "metabolic profiling", was defined by Horning and Horning (1971a, 1971b) as "multicomponent GC analyses that define or describe metabolic patterns for a group of metabolically or analytically related metabolites". The Hornings also suggested that "profiles may prove to be useful for charac- terizing both normal and pathologic states, for studies of drug metabolism, and for human development studies." Although metabolic profiling of biological mixtures is a relatively new technology, the analytical techniques that are used are not. These include paper chromatography, thin layer chromatography, column chromatography, gas chromatog- raphy (GC) and gas chromatography/mass spectrometry (GC/MS). 17 Of these techniques the most versatile and useful is GC/MS with computer assisted analysis of the data. Indeed, most of the ongoing research in the area of metabolic. profiling presently uses the technique of reconstructing mass chromatograms by computer from arrays of complete mass spectra (Hites and Biemann, 1970). Briefly, this technique involves the taking of complete mass spectra repetitively throughout an entire 60 analysis with subsequent computer storage of the data. The resulting information is a three dimensional array with axes of time, mass, and ion intensity. A plot of ion intensity vs. scan number for any particular mass is called a mass chromatogram. Examples of mass chromatograms can be seen in Figure 1. By examining the mass chromatogram of certain key ions, qualitative and quantitative information about specific compounds can be ascertained. Although most of the original work in the area of metabolic profiling was done using packed column GC separation, advances in capillary column technology in recent years, particularly in the area of column fabrication and coating techniques, have made this method very appealing as a separation vehicle for profiling analysis. Indeed, once experimental parameters are determined and stringently reproduced (i.e., sample preparation and GC parameters), capillary technology offers not only excellent qualitative but satisfactory quantitative analysis of complex mixtures. MSSMET. During the past 5-6 years, a system has been developed at the MSU/NIH Mass Spectrometry Facility for 18 Figure 1. GC/MS/DS analyses of urinary steroids from a normal female. A mixture of straight-chain saturated alkanes (C-20,22,24,26,28,30,32, 34) has been co-injected for calculation of methylene unit retention indicies. Mass chromatogram m/z+=85 shows where the various alkanes elute (top trace). The region around androsterone and etiochol- anolone is expanded in the bottom panels, and selected ion chromatograms for andro- sterone and etiocholanolone shown. Relative Intensity Relative Intensity Total Ion Intensity l I""U'"'r'"'l""l""If"'"_'v""f'1"V'f'f' 100 300 400 Sean Number ‘ J + m/e = 360 Androsterone Etiocholanolone m/e = 270 . 1 Total Ion Intensity '1 7 "v'U""""'""'U"WU"'vUr'r"""I""U""Uf 100 150 Scan Number 20 automated simultaneous qualitative and quantitative anlaysis of complex organic mixtures by GC/MS/DS systems (Gates gt _1., 1976a, 1976b, 1978a, 1978b, 1978c; Sweeley gt_g1., 1977; Gates and Sweeley, 1978; Sweeley, 1979). This technique uses methylene unit retention indices for the time dimension and an off-line reverse library search of the data obtained from GC/MS runs for qualitative and quantitative analysis of complex biological mixtures. The system is abbreviated as MSSMET (mass spectral metabolite search). Figure 1 illustrates the type of mass spectral data from which the MSSMET data system identifies and quantifies a particular compound in a complex mixture of chemically- similar substances. Data shown are for a GC/MS determina- tion of normal female urinary steroids. These data were obtained by repetitively scanning the mass spectrometer analyzer from 40 to 700 m/z every 4 seconds. The abscissa represents scan number and the ordinate represents ion intensity. The total ion intensity (TII) is the sum of the intensities of all ions recorded for any particular scan and is analogous to a GC trace. Also shown with the T11 is the mass chromatogram for m/z = 85 which indicates where the co-injected straight chain saturated hydrocarbons elute. The bottom of Figure 1 is an expanded display of the T11 between scan 70 and 175. Also shown are mass chromatograms for m/z = 270 and 360, which are prominent ions in the electron impact mass spectrum of the methoxime-trimethyl- silyl derivative of androsterone and etiocholanolone 21 (Table 6 lists the IUPAC name for trivial names and abbre- viations used). In determining the presence or absence of a particular compound listed in the MSSMET library, the computer looks for the presence of a GC peak of a character- istic ion ("designate ion") within a time "window". The exact location of the window is defined by the scan numbers at which the co-injected hydrocarbons elute. If a GC peak for the designate ion is found within this window, then the computer examines for the occurrence of other characteristic ions for this particular compound ("confirming ions") with BC peaks that maximize within 1 or 2 scans of the peak maxima for the designate ion. The program then integrates peak areas for each 60 peak found and calculates a correla- tion coefficient based upon the differences between the library and observed ratios of the designate and confirming ion intensities. If the correlation coefficient is greater than a value arbitrarily set by the operator, then the compound is considered "found" and relevant information concerning this compound (i.e., integrated area and peak height of the designate ion, retention index, and retention time of the compound, etc.) is stored in a "found-file". The correlation coefficient, peak detection and peak area integration algorithms are explained in greater detail later. Figure 2 shows a typical entry into the MSSMET compound "library" used by the computer to identify and quantitate compounds from a repetitive scanning GC/MS data file. MSSMET is a highly interactive program. The operator has a 22 Figure 2. Description of MSSMET library files. 23 KEY TO MSSMET LIBRARY* H OPN:/NM 135, OPN:/CB 69, *ENTRYTYPEzMSSMET NAM: * 1 ANDROSTERONE(3A-HYDROXY-5A—ANDROSTANE-17-ONE) TMI: 2529 DIN: 270 CIN: 270,999,360,546 0th“) *Typical library entry for MSSMET analysis of urine samples, based on a reverse library search using GC reten- tion indices and selected mass chromatograms. 1. Examples of options. MSSMET has 24 different options available to the user for interaction with the computer system. In the examples shown, the NM 135 option sets the "window" at 135 seconds around the retention index of a particular compound (androsterone, in this case). The computer will search all mass spectral data within the designated time frame for confirming ions of the selected compound. C0 is the value the correlation coefficient must equal or exceed for a particular compound to be printed out by the computer as a candidate. 2. Identifying compound number and name (common names and IUPAC). 3. Retention index (TMI). The retention index of androsterone, 2529, indicates that androsterone elutes between C-25 and C-26 (straight chain saturated hydrocar- bons) at a time after the elution of C-25 that is 29% of the time interval between the elution of C-25 and C-26. 4. The designate ion in this case is at m/z 270. This ion is usually one of the more intense ions in the mass spectrum of androsterone and is also an ion that does not occur in the mass spectra of compounds that elute near the compound in question, if possible. The designate ion is also used for quantitation. 5. "CIN" refers to the confirming ion set. This set includes the designate ion (peak at m/z 270); the intensity of the designate ion is reported as 999 in this set. Thus, in this example the confirming ion set consists of two peaks at m/z 270 and 360 having relative intensities of 99.9% and 54.6%, respectively; this information is represented above by the sequence "CIN: 270, 999, 360, 546". The number of confirming ions used can be selected by the operator up to 8. 24 number of decisions to make when building a library that influence MSSMET analysis of a particular data set. These decisions include the choice of ions for each particular entry into the library and of values for various options that influence the analysis of the data. In the MSSMET library, a retention index and key ions with their relative intensities are listed for each compound. One ion, the "designate ion", is used for quantitation. and the other ions are used to confirm identification of the compound. In the example shown, the compound in question, androsterone, has a retention index of 2529 and key ions are m/z = 270 and 360, with the intensity of m/z = 360 equal to 54.6% the intensity of m/z = 270. M/z = 270 has been chosen as the designate ion. Figure 3 shows a typical entry into a file generated by the MSSMET program which lists all the compounds found by the computer. The two most important pieces of information contained in the found file are the integrated areas of the designate ions for each compound and the correlation coef- ficient. In the example shown in Figure 3, androsterone has been identified with a correlation coefficient of 98 (100 is perfect) with the area of the designate ion being 43,317 (no units). In general, areas less than 1000 should be ques- tioned. Also shown is the amount of androsterone present in relationship to the amount of an internal standard (comment number 8 in legend to Figure 3). This value is calculated by dividing the area of the designate ion of androsterone by 25 Figure 3. Description of found file. The found file lists those compounds identified by the computer as being present in the sample. 26 KEY TO TYPICAL "FOUND" FILE 1 2 3 4 5 6 7 8 9 10 * 1 ANDROSTERONE 5 + 98 43317 O.177E+OO 21:53 - 0.09 + 99 20042 0.218E+00 PER DROP 16 17 18 19 20 11 12 13 14 15 2524 -5 134 137 141 MISSED 0 of 2 21 a) The "found" file lists those compounds identified by the computer as being present in the sample. The following information is printed with each found file. 1. Off-scale indicator. Indicates when maximum peak height is above a designated value. 2. Compound identification number. 3. Compound name. 4. Number of the peak out of number of candidate peaks found in time window. This number represents the chronolog- ical order of a series of chromatographic peaks within the designated time window for androsterone detected by the computer during data analysis. 5. Match category for peak area as determined by the correlation coefficient and the difference between the actual and the calculated retention index. Match categories include + (high correlation coefficient), (intermediate range correlation coefficient) and - (poor match with library retention index). 6. Correlation coefficient for peak area. 7. Peak area of designate ion. 8. Relative amount of compound using peak areas (area of designate ion relative to the area of the designate ion of the internal standard, corrected for the amount of internal standard added, ml of urine used and mg/ml of crea- tine). Value is unitless. The letter E indicates that the value is written in exponential form (i.e., the value shown is 0.177). Values of t 00 are nearly always significant. Depending upon the amount of internal standard added, values of E-02 or less could also be significant. 9. Retention time (minutes:seconds). 10. Difference of retention time from that predicted by library (minutes:seconds). 11. Retention index (methylene units). 12. Difference of retention index from library value. 13. Starting scan number of peak. 27 Figure 3 (continued) 14. Scan number at apex of peak. 15. Ending scan number of peak. 16. Match category for peak height. Match category defined in comment 5. 17. Correlation coefficient for peak height. 18. Peak height of designate ion. 19. Relative amount of compound using peak height for quantitation (see comment 8). 20. Method of area calculation. 21. Number of confirming ions missed. 28 the area of the designate ion of the internal standard, correcting for the amount of internal standard added, the ml of urine used, and the mg/ml of creatine. This value is referred to as the "area internal standard". After computer generation of a "found" file, MSSMET evaluates the data for quantitation purposes and prints this data in a "normalized found" file. A typical entry into this “normalized found" file is seen in Figure 4. The most important pieces of information contained in this file are the "area partial sum" and the "area internal standard" values. The "area internal standard" is calculated by dividing the integrated area of the designate ion for a particular compound by the integrated area of the designate ion for the internal standard. The "area partial sum" is calculated by dividing the integrated area of the designate ion for a particular compound by the sum of the integrated areas for the designate ions of all compounds found and multiplying this value by 10,000. Values for the "area partial sum" generally fall between 1 and 10,000. Values less than 1 should be questioned. Values for the "area internal standard" are entirely dependent upon the amount of internal standard added. Figure 5 shows part of a typical quantitative printout that is obtained for a typical human urinary steroid profile. In the example shown, the first line identifies the date of the computer analysis (March 10th, 1981), the name of the GC/MS data file (111118001.MSF), and the library 29 KEY TO NORMALIZED FOUND FILE* 1 2424 -5 ANDROSTERONE 98 281.445 0.117 99 317.620 0.218 2 2541 -6 ETIOCHOLANOLONE 99 380.289 0.239 99 395.004 0.271 3 2585 O DHA 91 11.136 0.007 84 8.637 0.006 1 3 3 4 5 6 7 8 9 10 *The normalized found file contains quantitative information for those compounds listed in the found file. The following information is printed with each normalized found file. Compound identification number. Retention index. Difference of retention index from library value. Compound name. Correlation coefficient for peak area. . Area partial sum. This value is calculated by dividing the integrated area of the designate ion for a particular compound by the sum of all integrated areas for the designate ions of all compounds found and multiplying this value by 10,000. 7. Height partial sum. Calculated in same manner as area partial sum but using peak heights instead. 8. Correlation coefficient using peak height. 9. Area internal standard. This value is calculated by dividing the integrated area of the designate ion for a particular compound by the integrated area of the designate ion for the internal standard. 10. Height internal standard. Calculated in the same manner as area internal standard but using peak heights of designate ion current responses. 0501-9me Figure 4. Description of the normalized found file. The normalized found file contains quantitative information on those compounds listed in the found file. Figure 5. 30 Example of a typical MSSMET quantitative printout (Found File) obtained for a normal female human urinary steroid sample. Format is the same as described in Figure 4. Information relative to the sample and date(s) of analyses are contained in each printout. Various unknown compounds (designated as UNI, UN2, ... etc.) have also been identified by their characteristic retention indices and ion currents. ooo o omm _o on oon m m_m no no ..uzr<¢nunsouoe o olom on . ooo.mm ooo mom mo ooo a. mno omm oo om.lllmz1xooo>xxomo>xHo-x-<:o4xomo>z<¢nunl"m.xxxomo>xumu o ooom om . ooo n own «o on omm m nmo me On .mzxomo>rxozo>rx_mnl.m.xxozo>r~mn-.~ r<¢nmn o anon m . omm o moo o on nmm o men n mm ~o~1>o. 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Some of the peaks have been labeled (see Figure legend). The top of Fig. 12 shows the region between C-24 and C-30. Characteristic mass chromatograms of the co-injected hydro- carbons, androsterone, etiocholanolone, pregnanediol, preg- nanetriol and 3o,17o-21-trihydroxy-Sa-pregnane-ll,ZO-dione (THE) are displayed. It is important to understand that these steroids were identified not only by their character- istic ion currents, but also by their characteristic reten- tion indices. The bottom of Fig. 12 shows the set of ions for preg- nanetriol that the MSSMET program used to identify and quan- titate this compound. For each compound searched for by the MSSMET program, one ion that is both characteristic and intense was used for quantitative purposes (the designate ion) and the presence and relative intensities of a few other ions produced during fragmentation were used to confirm the identity of the compound in question (confirming ion). Retention index as well as good ion statistics on the designate and confirming ions are crucial to the identifica- tion of each compound. In the example shown, m/z 255 is used to quantitate pregnanetriol and the other peak area intensities were used in the calculation of a confidence coefficient indicating the presence of this compound. Reproducibility of the computer-assisted automated meta- bolic profiling analysis of urinary steroids using capillary Figure 12. 65 GC/MS analysis of urinary steroids using a 50 m 0V-101 WCOT fused silica capillary column. Shown are characteristic mass chromatograms for the co-injected hydrocarbons, androsterone, etiocholano- lone, pregnanediol, pregnanetriol and THE. The bottom graph shows the set of ions for pregnanetriol that the MSSMET program uses to identify and quantitate this compound. Conditions of analyses are given in the text. A K-factor (Gates gt g1., 1978b, 1978c) was determined for pregnanetriol and the amount injected was calculated to be 18 ng. : ml: 488 THE 1 l : ml: 270 Androsterone and 1 Etiocholanolone V Y V V VA A 4 I mIz 255 Pregnanetriol ml: 117 Pregnanodlol J c-zc c-zo c-za . mlz 05 C-30 l 7 V 7' V V 7* l 1" \T "TV' '— 800 1000 1” 1200 1400 scan woman \ / \ / : Pregnanetriol ml: 436 1 4 1 1 ml: 255 q d 3 ml: 245 v a- i q : mlzZJI : A M : mlzZIS 4 4 4 A A 1230 1240 1250 1260 SCAN NUMIER 67 column GC/MS is summarized in Table 2. Values are the integrated peak areas determined by the MSSMET program of a characteristic ion for each steroid expressed as a percent of the sum of the areas for these five urinary steroids. Data are from four separate GC/MS analyses of the same sample using a 25-m SP-2100 WCOT fused silica capillary column (0.2 mm 1.0.). Data are given for the earlier elut- ing steroids since their GC peaks are much sharper than those occurring later in the GC run (the cortols and the cortolones, for example) and thus they are the most diffi- cult to quantitate accurately by the repetitive scanning technique. The overall precision was 4.8% for these four separate GC/MS analyses (calculated by expressing the stan- dard deviation as a percent of the mean and averaging these values for the five steroids shown). Also shown in Table 2 is the ratio of etiocholanolone to androsterone as deter- mined by MSSMET. Using a K-factor (calculated by knowing the ratio of the intensities of ion currents for the internal standard, cholesteryl butyrate, and pregnanetriol for equal amounts of each compound [Gates gt g1., 1978b, 1978c)), the amount of pregnanetriol was calculated to be 18 ng injected (150 ng/ml urine). These results demonstrate that a 2-sec scan cycle time is sufficient to quantitate urinary steroids by capillary column GC/MS using recon- structed mass chromatograms from repetitive scanning data. 68 Table 2. Precision: Capillary Column GC/MS Profiling of Urinary Steroidsa R u n N u m b e r Steroid 1 2 3 4 Mean : 5.0.b Androsterone 9.4 8.6 10 10 9.50 i 0.7 Etiocholanolone 12 12 ’13 13 12.5 1 0.6 Pregnanediol 59 59 57 56 57.8 i 1.5 PregnanetriolC 14 16 15 15 15.0 t 0.8 THE 5.4 5.8 5.8 5.9 5.73 e 0.22 Etio./Andro.d 1.2 1.3 1.3 1.3 1.28 1 0.05 aValues are the integrated peak areas determined by MSSMET of a characteristic ion for each steroid expressed as a percent of the sum of the areas for these 5 urinary steroids. Data are from 4 separate GC/MS analyses of the same sample using a 25 M SP-2100 WCOT fused silica capillary column (0.2 mm 1.0.). The average precision was t 4.8 percent (calculated by expressing each 5. D. as % of the mean for the 5 steroids shown). CA K- factor (Gates et al., 1978b,1978c) was determined for pregnanetriol and the_ amount injected corresponded to 18 ng. Ratio of etiocholanolone to androsterone. 69 Optimization of GC/MS conditions for automated reverse library search of selected mass chromatograms using capillary columns. We have found that two factors must be taken into consideration when developing optimal conditions for the quantitative analysis of complex mixtures by capil- lary column gas chromatography repetitive scanning mass spectrometry that are not considerations for capillary gas chromatography alone. First, capillary gas chromatographic peaks are much sharper than packed column peaks and require shorter scan cycle times to obtain an adequate number of data points for quantification. Thus, while sharp peaks are desirable for quantitative analysis of complex mixtures by capillary gas chromatography alone, this was detrimental when appropriate scan rates could not be realized with the instrument system available. The second important consider- ation was the detection and accurate quantification of minor components in a complex mixture. Compounds that were 100- 1000 times less concentrated than the major components in a complex mixture were quantified relatively easily by capil- lary gas chromatography with FID, as long as they were well separated from nearby peaks. However, when repetitive scan- ning GC/MS was used, sensitivity problems occurred. Figure 13 shows the urinary steroid metabolic profile of a normal adult male subject. Selected mass chromatograms for a number of the major urinary steroids have been plotted above the total ion intensity (see Figure legend). The capillary Figure 13. 70 Urinary steroid metabolic profile of a normal adult male subject. The total ion intensity is plotted against scan number (2s scan cycle) at the bottom and selected mass chromatograms of designate ions for a few major steroids are plotted above. Labelled peaks are (1) THE, (2) THF, (3) allo-THF, (4) cortolone, (5) a-cortolone, (6) Ila-hydroxyandrosterone, (7) 1IB-hydroxyetiocholanolone, (8) internal standard, (9) Cholesterylbutyrate, added as a second internal standard, (10) androsterone, (11) etiocholanolone, (12) DHEA, (13) pregnanetriol (14) pregnanediol. Conditions of analysis are as follows: LKB-2091 GC/MS with a dual Digital Equip- ment Corp. POP-8e based foreground- background data system (Martin gt al., 1980); 25 M OV-1OI fused silica capTllary column, 0.32 mm 1.0.; p = 25 cm 5' ; split injection, 2 pl of sample injected; gas chromatographic oven temperature progfamming from 180 to 280°C at 2°C min' ; El ionization at 70 eV; 2 s scan cycle interval, m/z 50-550. . 11 488 1 2‘3 472 ‘1: “9 : 111 1- r - . - , i 6H7 448 4 7 435 a 8 A a 1 1 e 1 1111 1111111 l'll 111111 13 255 1111111 I14 117 Total Ion Intensity fiY‘rYYVVVIYrV—YIYY'Y'VVYf'IVIVIVYYY'YYUVIfY‘YIYYVV‘YYVV—I—TVW 500 1000 1500 SCAN NUMBER 72 gas chromatograph had to be overloaded with sample when using repetitive scanning mass spectrometry for detection and analysis of urinary steroids. The lower limit of accurate quantification for any compound by mass chromatography from repetitive scanning GC/MS data is is determined by such factors as the percent total ionization of the ion used for quantification of a particular compound, scan speed of the mass spectrometer, cleanliness of the source and other factors. For full mass range repetitive fast scanning GC/MS, this limit is general- ly in the range of 1.0-10 ng injected for modern instruments operated in an ordinary manner. Thus, if minor components are 100-200 times lower in level than the major components of a mixture, several hundred nanograms of the major compo- nents must be injected for accurate quantification of these minor components. Unfortunately, this resulted in overload- ing of those capillary columns that offered sufficient column efficiency to separate steroid stereoisomers. Although this was not aesthetically pleasing, this condition did not present an analytical problem so long as compounds with virtually identical mass spectra were still chromato- graphically resolved. A moderate degree of overloading of the capillary column therefore provided a wider concentra- tion dynamic range than would otherwise have been possible. Optimal metabolic profiling analysis of urinary steroids requires determination of conditions which allow adequate chromatographic separation of major stereoisomers such as 73 androsterone and eticholanolone, THF and allo-THF. Although these peaks were somewhat distorted by overloading, the precision of quantification was not compromised so long as the chromatographic separation of these epimers was maintained. These considerations are graphically illustrated in Figures 14, 15 and 16. Figure 14 shows the change in peak shape and the increase in number of data points across a peak that occur with increasing amounts of hydrocarbon, n-pentadecane, injected onto the capillary column. The exact sample size where a particular capillary column will become 'Overloaded' is related to the internal diameter of the capillary column and the stationary phase film thickness. With capillary columns that offer sufficient performance to separate steroid stereoisomers we have observed that steroids overloaded when between 100 ng and 500 ng Of each steroid are injected. Figure 15 shows total ion intensity v. scan number in the region where the internal standard elutes. Also shown are characteristic reconstructed mass chromatograms for one of the internal standards (m/z 435), 118-hydroxyandrosterone (m/z 448; first peak) and 118-hydroxyethiocholanolone (m/z 448; second peak). Although there is a slight overlap of the two m/z 448 peaks and the m/z 453 peak exhibits a moderate degree of 'fronting', each separate profile is well defined and the peak areas can be accurately integrated using a fully automated computer search of selected mass chromatograms. Figure 14. 74 Changes in peak shape and number of points across a gas chromatographic peak following increasing amounts of n-pentadecane injected into a 25 m 0V-101 fused silica capillary column (0.32 mm 1.0., 150°C isothermal). m/z 99 and 85 are plotted v. scan number for 5, 50, 200, 350, 600 and 2000 ng injected. A 2 s scan cycle was used. 0.08 coon .03 N £25050». 0 com p :9. SE «0.0 £83.00 32.33 3:? coma“. 57>O E mu O: OOON ON - OFF 2 n 8 K mm . 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F can econoamoepchoegx . m w w firIrIY wvv Figure 16. 78 Urinary steroid metabolic profiles using a 25 m 0V-101 fused silica capillary column with FID (top trace) and the same column with MS detection (repetitive scanning; 2 s scan cycle). In the top trace THF and allo-THF are labeled to demonstrate the complete separation of these epimers using FID. The bottom trace shows a GC/MS analysis using the same column. A 2 second scan cycle was used. 25 meter 0V-lOl fused scha caleary cohlmn HP 5840A with F l D NdoJHF mile i l' 25 meter 0V-101 fused silica capillary J LKB-209l, so-eoo mlz, 2 sec. cycle “472 l «T 7 THF -4 I l lALLO-THF i 4 l "'I'"! g 3: i l ? {H l '. m -..... . , .-—--- - ... ...— -~ .. W a: E: 2;... J i illqhizkg'li‘lqu l M M' l ’ N ‘ 'l 5 ' 'T—T—T’T-TTTT—T‘V—T—T—T—T‘V—TTTT Y TTTT‘T‘fT—T‘T T_TT:F#7‘# 80 The major steroid epimers that were the most difficult to separate were THF and allo-THF. The top trace of Figure 16 shows a urinary steroid metabolic profile obtained using a 25 m, 0.32 mm 1.0., 0V-101 fused silica capillary column with FID. THF and allo-THF are labeled to demonstrate the complete separation of these epimers. The bottom trace in Figure 16 shows a GC/MS analysis using the same column (T11 is plotted against scan number). The column has been over- loaded such that these epimers are no longer completely separated. However, the degree of separation is such that each epimer can still be independently quantitated well within the precision of the overall method (separation shown by mass chromatogram m/z = 472; insert). It might be informative to consider further some of the advantages and disadvantages of packed and capillary columns for automated metabolic profiling analysis of urinary steroids by a GC/MS data system using reverse library search of selected mass chromatograms. Capillary columns are reported to offer several distinct advantages over packed columns, such as increased resolution, decreased sample loss due to adsorption during chromatography (Axelson and Sjbvall, 1977), and potentially shorter analysis times. However, accurate quantification of minor components in complex mixtures by repetitive scanning GC/MS using capil- lary column chromatography requires high sensitivitiy and fast scan speeds to establish a sufficient number of data points in reconstructed mass chromatograms. Quadrupole mass 81 filters offer sufficiently short scan cycle times but exhibit a severe mass intensity discrimination problem above approximately m/z 500, where many of the qualitatively characteristic fragment ions of methoxime-trimethylsilyl derivatives of steroids occur. Although magnetic sector instruments do not have this problem they have the distinct disadvantage of limitations in the repetitive scan cycle times. It was our experience that for many applications, packed column gas chromatography was adequate for metabolic profil- ing analysis of urinary steroids and was, in fact, quite superior in certain situations where differences in the levels of minor steroid metabolites were of interest. As an example, in the studies described in sections III and IV, certain differences that were detected by packed column GC/MS data systems were not observed by capillary column GC/MS data systems unless the columns were severely over- loaded. Thus, in certain situations (metabolic profiling of body fluids, for example) it would be prudent to use both packed and capillary columns, taking advantage of the qualities each of these systems offers for optimal qualita- tive and quantitative analysis. The investigations just described have led to the development of an automated GC/MS/DS procedure and a capil- lary column GC/FID procedure for reproducible quantitative analysis of complex mixtures of steroids. Important features of the GC/MS/DS system are the use of both packed 82 and capillary column chromatography, a non-mass discriminat- ing mass analyzer and a fully automated reverse library search procedure using methylene unit retention indices and reconstructed mass chromatograms. SECTION III Animal Studies SECTION III INTRODUCTION This section describes experiments, using the rat as a model, designed to examine the effects of PBBs on the urinary steroid metabolic profile, and to correlate differences in the profile with known specific effects these chemicals have on steroid metabolism 13 11332 (i.e., increased 63, 73 and/or 16a-hydroxylations; see background in Section I). Since steroid metabolism and excretion in the rat is very complex and qualitatively different from man, an overview of the findings of the most relevant investigations into steroid hormone metabolism and excretion in rats is presented below. Metabolism of steroid hormones in rats. Steroid hormones must be metabolized to more polar molecules in order to be excreted in the urine and feces. In general, this is accomplished by a series of oxidative, reductive and conjugation reactions in both man and rats. Although man and the rat have certain metabolic reactions in common, there are many species differences (see Introduction, Section IV, for a detailed discussion of steroid metabolism in man). For example, while both man and rats reduce the 4-ene-3-one A-ring configuration, the rat has extensive 83 84 further metabolism that occurs via gut microbes during enterohepatic circulation (Eriksson, 1971a). Some of the microbial metabolites are reabsorbed and may be excreted in the urine (Gustafsson, 1968; Eriksson and Gustafsson, 1970; Gustafsson, 1970) as well as the faeces. Also, there are marked sexual differences in the metabolism and excretion patterns of steroids which are in part due to differences in the oxidative metabolism of steroids in the livers of male and female rats as well as to differences in conjugation reactions (Eriksson gt_gl., 1972). Lisboa gt 31. (1968) showed that rat liver microsomes are capable of hydroxylation of testosterone to 3-keto-A4- hydroxy metabolites. These metabolites were identified as 23-, 63-, 7a-, and 16a-hydroxytestosterone and 63,16a,17e- trihydroxyandrost-4-ene-3-one. Gustafsson £5 al. (1968a) reported that saturated C-19 steroids with a 3-keto-5a- configuration were formed from rat liver microsomes. These metabolites were reported to be 25-, 63-, 7a- and 16a- hydroxy-Sa-dihydrotestosterone. These compounds were also formed from 23-, 68-, 7a- and 16a-hydroxytestosterone precursors. When Sa-dihydrotestosterone was used as sub- strate, 23-, 7a- and 6a-derivatives were formed but not 63- hydroxy-Sa-dihydrotestosterone. Gustafsson gt al. (1968b) also reported the formation of several Sa-androstanetriols by rat liver microsomes. When testosterone was used as a substrate, Sa-androstane-Za,3a,173-triol, Sa-androstane-3a, 7a,173-triol and Sa-androstane-3a(and 33),16a,17e-triol were 85 formed. 23- and 7a-Hydroxytestosterone precursors gave androstane triols with a 3a-configuration while incubation with 63- and 16a-hydroxytestosterone gave both 30- and 33- epimers. When Sa-dihydrotestosterone was used as a substrate, Sa-androstane-23,3a(and 33),173-triol, Sa-andro- stane-3a(and 33),7a,173-triol and Sa-androstane-3a(and 33), 16a,173-triol were formed (no 63-hydroxy steroids were formed). Only 5a-androstane-23,3a,173-triol was formed when Sa-androstane-Ba,173-diol was incubated. Sa-Androstane-33, 173-diol was not hydroxylated by the microsomal preparation. 6a-Hydroxylation of testosterone and androstenedione by rat liver microsomes has also been reported (Gustafsson and Lisboa, 1970a). Several metabolites of 4-androstene-3,17- dione, Sa-androstane-3,17-dione, 3a-hydroxy-Sa-androstane- 17-one and 33-hydroxy-Sa-androstane-17-one are formed in rat liver microsomes. These include 3a(and 33),16a-dihydroxy- Sa-androstane-17-one, 3a(and 33),7a-dihydroxy-Sa-androstane- 17-one, 33,73-dihydroxy-Sa-androstane-17-one and 33,173- dihydroxy-Sa-androstane-16-one (Gustafsson and Lisboa, 1970b). 6a- and 63-Hydroxylation of testosterone has also been reported to occur in rat testis preparations (Gustafs- son and Lisboa, 1970c). A number of metabolites of testos- terone have been identified in the feces of germ-free male and female rats. These include 3a,113-dihydroxy-Sa-andro- stane-17-one, 3a,7a-dihydroxy-Sa-androstane-17-one, 3a,15a- dihydroxy-Sa-androstane-17-one, Sa-androstane-3a,5a, 173- triol and Sa-androstane-Ba,173-diol (Gustafsson and Sjovall, 1968). 86 Differences have been noted between male and female rats in the metabolism of corticosterone by the isolated perfused liver preparation. Metabolites in the male rats were 5a- pregnane-Ba,113,203(and 20a),21-tetrol and Sa-pregnane-33, 113,203,21-tetrol. Only small amounts of 5a-pregnane-Ba, 113,203,21-tetrol were produced in female rat liver while the predominant metabolites were 3a(and 33),113,15a,21- tetrahydroxy-S3-pregnane-20-one and 3a(and 33),113,21- trihydroxy-Sa-pregnane-ZO-one. Female rat livers also produced far greater amounts of mono- and disulfate conju- gates than did the male rat livers (Eriksson and Gustafsson, 1971). Gustafsson and Sjovall (1968b) identified a number of 15a- and 21-hydroxylated C-21 steroids in germ-free rats that were not found in conventional rats. 3a,118,15a,21- tetrahydroxy-Sa-pregnane-ZO-one and 3a,15e,21-trihydroxy-5a- pregnane-11,20-dione were the major peaks while smaller amounts of 113,Zl-dihydroxy-Sa-pregnane-3,20-dione, 3a,113, 21-trihydroxy-Sa-pregnane-ZO-one, 3a,21-dihydroxy-5a- pregnane-11,20-dione and 3a,16a-dihydroxy-5a-pregnane-ZO-one were found. The absence of 21-hydroxylated steroids in feces from conventional rats has been explained by 21-dehydroxylation of 21-hydroxy-20-keto steroids by gut microbes (Eriksson 33 31., 1969). Rat liver microsomes can hydroxylate progesterone at a number of positions. Gustafsson and Lisboa (1970d) reported the formation of 2a-, 63-, 6a-, 7a-, 15a-, 153- and 16a- hydroxyprogesterone in rat liver microsomes. Several 87 hydroxypregnanolones have been isolated from the feces of male and female germ-free rats. These include 3a,16a- dihydroxy-Sa-pregnane-ZO-one, 33,16a-dihydroxy-S-pregnene- 20-one and 33,16a-dihydroxy-Sa-pregnane-ZO-one (Gustafsson £3 al., 1968a). When [4-14C]pregnenolone was injected intraperitoneally in female rats, 70% of the radioactivity was recovered in the bile (via cannula), with 45% of this radioactivity being in the monosulfate fraction, 28% in the disulfate fraction, 22% in the glucuronide fraction and 2% in the free fraction (Cronholm £3 31., 1971). Metabolites of pregnenolone in the glucuronide fraction included 3a- hydroxy-Sa-androstane-17-one, 3a-hydroxy-5a,173-pregnane-20- one and 3a,16a-dihydroxy-Sa-pregnane-ZO-one while 3a,7a- dihydroxy-Sa-androstane-17-one, 3a,1Sa-dihydroxy-Sa-andro- stane-17-one, 3a,113-dihydroxy-Sa-androstane-17-one and 3a, 163-dihydroxy-Sa-pregnane-ZO-one were found in the monosul- fate fraction and 3a,7a-dihydroxy-Sa-androstane-l7-one, 3a, 113-dihydroxy-Sa-androstane-l7-one, Sa-pregnane-33,203-diol and 3a,21-dihydroxy-Sa-pregnane-ZO-one were found in the disulfate fraction. In these experiments the quantitatively most important steroid metabolites were the unlabeled corti- costerone metabolites 3a(and 33),113,21-trihydroxy-5a- pregnane-ZO-one and 3a,113,15a,21-tetrahydroxy-5a-pregnane- 20-one. Following intraperitoneal administration of [4-14C] pregnenolone and [4-14C]corticosterone to female germ-free rats the following steroids were recovered in the 88 feces: disulfate fraction; 3a(and 3B),118,21-trihydroxy-Sa- pregnane-ZO-one, monosulfate fraction; 38,17a-dihydroxy-5a- pregnane-ZO-one, 5a-pregnane-3a,16a,20a-triol, 2a,33,16a- trihydroxy-Sa-pregnane-ZO-one and 3a,158,16o-trihydroxy-5a- pregnane-ZO-one (Eriksson and Gustafsson, 1970b). Conven- tional rats showed a different distribution of radioactivi- ty, in part due to the formation of 38,11o-dihydroxy-5a- pregnane-20-one and 17a-pregnane derivatives of some of the above metabolites. The steroids identified in the feces of conventional rats were estriol, 3a,15a(and ISBl-dihydroxy- 5a,17a(and 17B)-pregnane-20-one, 3o,19-dihydroxy-5o,17a- pregnane-ZO-one, So-pregnane-Ba,16a,20-triol, 38,11a- dihydroxy-Sa-pregnane-ZO-one, 2a,3a-dihydroxy-5a,17a- pregnane-ZO-one and 3a,17o-dihydroxy-5a-pregnane-ZO-one. In a subsequent experiment using the same protocol a large number of steroids were noted in the free fraction in conventional rats while only a small number could be found in the free fractions of germ-free rats (Eriksson, 1970). In the conventional rats, 3o,208-dihydroxy-Sa-pregnane-11- one and 38,208-dihydroxy-Sa-pregnane-ll-one were charac- terized as metabolites of corticosterone whereas 30-hydroxy- 5a,1fl:-pregnane-20-one, 3a-hydroxy-5a,173-pregnane-20-one, 38-hydroxy-5a,17a-pregnane-20-one, 33,153-dihydroxy-5a- pregnane-ZO-one, 33,15a-dihydroxy-5a-pregnane-ZO-one and 3a, 16a-dihydroxy-Sa-pregnane-20-one were identified as metabo- lites of pregnanolone. No steroids were completely identified in the germ-free animals. Sulphohydrolase and 89 glucuronohydrolase activities have been characterized in the intestine of conventional and germ-free rats. The sulpho- hydrolase activity was present only in conventional rats (glucuronohydrolase activity being present in conventional and germ-free animals) and could hydrolyze sulfuric acid esters of the 3a.33,173 and 21-hydroxyl groups (Eriksson and Gustafsson, 1970c). Eriksson 33 31. (1971) reported the presence of eight 15-hydroxylated C-21, 0-4 steroids in urine and feces of conventional female rats: 3a(and 33),113,15a-trihydroxy-Sa, 143-pregnane-20-one, 3a(and 33),1Sa-dihydroxy-5a,143-pregnane- 11,20-dione, 3a(and 33),15a,203-trihydroxy-5a,143-pregnane- 11-one and 5a,143-pregnane-Ba(and 33),113,15a,203-tetrol. These were shown to be microbial metabolites of 15a, 21- hydroxylated C-21, 0-5 steroids present in germ-free rats. The occurrence of steroid monosulfates in the urine of conventional and germ-free rats was investigated by Gustafsson (1970). Steroid monosulfates were not present in the urine of male rats, conventional or germ-free. Female germ-free rats excreted Sa-androstane-Ba,173-diol, 3a,113- dihydroxy-Sa-androstane-17-one, 3a,7a-dihydroxy-Sa- androstane-17-one, Sa-androstane-3a,7a,173-triol, 3a,16a- dihydroxy-5a-pregnane-20-one, 30,1Sa-dihydroxy-Sa-pregnane- 20-one, 3a(and 33),17a-dihydroxy-Sa-pregnane-ZO-one, 5a- pregnane-3a,16a,20a-triol, 113,21-dihydroxy-5a-pregnane- 3,20-dione, 3a,113,21-trihydroxy-Sa-pregnane-ZO-one, 3a,15a, 21-trihydroxy-Sa-pregnane-ll,20-dione and 3a,113,15a,21- tetrahydroxy-Sa-pregnane-ZO-one monosulfates. Conventional 90 female rats excreted 3a,19-dihydroxy-5a-androstane-17-one, 3a-hydroxy-5a,17a-pregnane-20-one, 3a-hydroxy-5a-pregnane- 20-one, 3a-hydroxy-Sa-pregn-16-ene-20-one, 3o,19-dihydroxy- 5a,17a-pregnane-20-one and 33,ISa-dihydroxy-Sa-pregnane-ZO- one monosulfates in addition to the above steroid monosul- fates present in the urine of germ-free rats. Eriksson (1971b) has examined steroids in the urine and feces of conventional and germ-free male rats derived from [4-14C] pregnenolone and [4-14C]corticosterone (intraperitoneal injection). In general, the metabolites found in both urine and feces were more polar than those derived from [4-146] pregnenolone and [4-14C]corticosterone in female rats. A series of Sa-pregnane-3,11,16,20,21-pentol isomers were the major metabolites of corticosterone in the urine and feces of conventional male rats. Sa-pregnane-3a(and 33),113,203, 21-tetrol and 3a(and 33),203-dihydroxy-Sa- pregnane-ll-one were excreted in the feces but not the urine. In the germ-free male rats Sa-pregnane-3a(and 33), 113,203,21-tetrols and 3a(and 33),113,21-trihydroxy-5a- pregnane-ZO-one were the predominant species in the mono— and disulfate fraction from feces although minor amounts of C-19 0-3 and C-21 0-3 steroids were found in the monosulfate fraction. In the germ-free male rats all radioactive steroids in the urine were in the free raction. These compounds were C-19 0-5, C-21 0-4, C-21 0-5, C-21 0-6 and C-21 0-7 steroids that could not be fully characterized. 91 In summary, although man and the rat have a number of metabolic pathways in common, there are a large number of differences. In general, metabolites of steroids in the urine and feces of rats (both conventional and germ-free) are more polar than steroid metabolites in the urine and feces of man. Enterohepatic circulation in the rat is more important for steroid metabolism than it is in man. Ring hydroxylations are an important (major) route of steroid metabolism in the rat with the 2,6,7,15,16, and 19 positions being the most important. Steroids with the 3-keto-4-ene configuration can undergo hydroxylation before reduction of the A-ring. Steroids are excreted unconjugated or as glucuronides, monosulfates or disulfates in rats, with marked sexual differences in the distribution of metabolites in these fractions. In addition, urinary steroids in rats reflect metabolism of corticosterone, the major adrenal steroid in rats, while urinary steroids in man reflect metabolism of cortisone and cortisol, the major adrenal steroids in that species. EXPERIMENTAL Materials. All materials were obtained from the sources previously described in Section II. Animals. Male and female Sprague-Dawley rats were purchased from Spartan Farms, Haslett, MI. Animals were housed four to a cage. A 12-hour day-night cycle was used. Isolation of Steroids. Steroids were isolated by the procedure described in Section 11. Gas Chromatography. Urinary steroid metabolic profiles were generated using a 60 m DB-l bonded phase fused silica capillary column as previously described in Section II. Gas Chromatography/Mass Spectrometry. Urinary steroid metabolic profiles were generated by GC/MS/DS as previously described in Section II. Collection of Urine Samples. Urine was collected using stainless steel metabolic cages. The collection mechanism allowed for separation of urine from feces. Urine was collected over a 24 hour period in a 50 ml Erlyenmeyer flask. Animals were allowed food (crushed) and water ad libitum during the collection period. 92 RESULTS Initial studies focused on determining the effect of exposure to PBBs on the urinary steroid metabolic profile. Urine was obtained from two groups of adult female rats (4 animals per group, pooled sample collection) that had been exposed to 100 ppm PBBs in the diet from day 8 of gestation until 4 months of age when urine samples were collected. Urine was also obtained from two groups of control animals. Urinary steroids were isolated using Method I (see Section II). Urinary steroid metabolic profiles were generated by packed column GC/MS/DS. These pilot studies indicated that the urinary steroid metabolic profiles in PBBs-exposed and non-exposed groups were qualitatively the same. However, certain quantitative differences were observed. These observations are illustrated in Figure 17 and Table 3. Figure 17 shows the urinary steroid metabolic profiles obtained for one of the PBBs-exposed groups and the profile obtained for one of the control groups. Scan number is plotted against m/z = 73, a characteristic fragment ion of trimethylsilyl derivatives. Qualitatively the two profiles are very similar. However, a number of quantitative differences, or apparent quantitative differences, can be seen, especially in the later region of the chromatogram. 93 Figure 17. 94 Urinary steroid metabolic profiles of female rats exposed to 100 ppm PBBs chronically (top trace) and of an apprOpriate control group. Urinary steroids were isolated using Method I as described in Section II, Development of Methods. Profiles were generated by GC/MS/DS. Scan number is plotted against m/z = 73, a characteristic fragment ion of TMS derivatives. Quantitative differences are seen in the later eluting compounds. 0.. h lAVIIOl’I . l ..Om....zco 7 : l- lllllll.-ll . mm mmn. 96 Table 3. Effect of Chronic Exposure to PBBs of Female Rat Urinary Steroids Peak Retention Number Index Control2 3883 3 2515 0.208 0.229 4 2568 6.67 14.6 6 2711 0.465 0.957 7 2797 0.615 1.52 9 2875 1.34 0.92 12 3017 1.69 18.4 14 3126 2.91 16.5 16 3331 2.05 14.5 1 Female rats were exposed to 100 ppm PBs from day 8 of gestation until 4 months of age when urine samples were collected. 2 Values are the average for 8 animals per group with 4 animals pooled per sample (n=2). Values were calculated using the following formula: P_A.P_ 1111.9). PACB b(ml)c(mg/ml) where, PAu = peak area of designate ion of compound in question. PACB = peak area of m/z=255 of cholesteryl butyrate. a = ug of cholesteryl butyrate added. b = ml of urine used. c = mg creatinine per ml of urine. 97 This is the region of the chromatogram where MO-TMS deriva- tives of metabolites of corticosterone, cortisol and cortisone elute (retention index 2900-3300). Data for some of the peaks observed have been tabulated in Table 3. Values are the average for the two groups (i.e., two control pooled collections and two PBBs pooled collections). Each value was calculated as the peak area of an intense and characteristic fragment ion for each compound in question, divided by the peak area of m/z 255 of cholesteryl butyrate, the internal standard, times the amount of internal standard added, divided by the mls of urine extracted and the creatinine concentration. Most peaks were approximately the same in the experimental and control groups, illustrated by peak numbers 3,4,6,7 and 9. However, certain compounds were very different. For example, data for peaks at retention index 3017, 3126 and 3331 are tabulated to show that some peaks in the steroid profiles were apparently different in the two groups. Mass spectral interpretation indicated these compounds were metabolites of corticosterone. Based on these preliminary results, the study was expanded to include a large enough number of subjects to make statistical comparisons between the P885 exposed and control groups. The treatment protocol of Dent gt g1. (1976) was chosen because the physiological and biochemical effects of P885 are well characterized with this regimen. Twenty one day-old female Sprague-Dawley rats were treated with a single 150 mg/kg I.P. dose of P885 at day 21 of age. 98 Urines were collected 8 days later over a 24-hour period. Four animals were housed per metabolic cage with four cages in each experimental group. Urinary steroids were isolated and derivatized by method I. Samples were analyzed both by capillary column GC and GC/MS/DS. Most GC peaks, determined by capillary column GC or GC/MS/DS, were not different between the control and P885 groups. Although no qualitative differences were noted, certain peaks were quantitatively different. Data for those peaks where mass spectral interpretation was informative are presented below. The quantitative differences occurred in the "corticosteroid" region of the chromatogram and are given in Table 4 and Figures 18, 19 and 20. Values shown in Table 4 were calculated by dividing the peak area of a char- acteristic mass chromatogram for the compound in question by a characteristic mass chromatogram for the internal standard, cholesteryl butyrate, times the amount of internal standard added, divided by the mls of urine extracted and the concen- tration of creatinine. The group means t 5.0. are shown on the right. Control and P885 data were compared by Student's t-test and found to be significantly different at p<0.01. Figure 18 shows the mass spectrum for the first peak listed in Table 4. This is a typical mass spectrum for the MO-TMS derivative of a tetrahydroxy-pregnane-one. The molecular ion (m/z=683) and M+-31 (m/z=652) are usually observed with these derivatives. Also seen are character- istic losses of M+-90-31, M+-2X90-31 and M+-3x90-31. 99 Table 4. Changes in urinary excretion of some corticosterone metabolites in female rats following acute exposure to PBBs. Amountb X i 8.0. C A G E N U M B E R 1 2 3 4 1) C2105C Control 4.4 3.5 4.0 0.9 3.2 i 1.6 PBBs 15.9 8.0 7.6 10.6 10.5 t 3.8* 2) 021050 . Control 1.0 2.4 2.8 2.2 2.1 e 0.8 PBBs 0.64 0.41 0.26 0.25 0.39 t 0.18* 3) c2105c Control 2.3 1.4 1.6 0.3 1.4 2 0.83 PBBs 6.7 3.6 7.0 3.7 5.3 t 1.85* 4) 02106d Control 1.7 1.8 3.8 3.2 2.6 e 1.0 PBBs 0.88 0.79 0.48 0.50 0.66 e 0.2* a 150 mg/kg i.p. to 21 day old female Sprague-Dawley rats. Urines were collected 8 days later. 16 animals per group. Urine samples pooled from 4 animals (n=4). 9 For numbers 1,2 and 3, the value is the peak area of m/z 472 for the C2105 steroid divided by the peak area of m/z 255 of the internal standard, cholesteryl butyrate, times the amount of internal standard added, divided by the mls of urine extracted and the concentration of creatinine. For number 4 the value is the peak area determined by capillary column GC divided by the peak area of the internal standard, cholesteryl butyrate, times the amount of internal standard added, divided by the mls of urine extracted and the concentration of creatinine. C A tetrahydroxy-pregnane-one. d A pentahydroxy-pregnane-one. *Significantly different from the control by Student's t-test, p<0.01. Figure 18. 100 Mass spectrum of the MO-TMS derivative of a tetrahydroxy-pregnane-one that increased in the urine of P885 exposed female rats. The molecular ion (m/z=683) and MT-31 (m/z=652) are observed, as are characteristic losses of MT-90-31, M+-2X90-31 and MT-3X90-31. Likely structures for this steroid are 36,113,156(or 166),21-tetrahydroxy-Sa- pregnane-ZO-one or 3a,63,113,156(0r 16o)- tetrahydroxy-Sa-pregnane-ZD-one. ooo +2 AHM-+ZV Afim-om-+zv . mmm mmm mmm . com co: Afim-omxm-+zv Afim-omxm-+zv NR: «mm com - o.N o.H l ax mwa Figure 19. 102 Mass spectrum of the MO-TMS derivative of a tetrahydroxy-pregnane-one that decreased in the urine of P885 exposed female rats. The molecular ion (m/z=683) and M+-31 (m/z=652) are observed, as are characteristic losses of M+-90-31, M+-2X90-31 and M+-2X90-31. The ion at m/z=170 is characteristic of M0-TMS derivatives of C-15 hydroxy, C-20 keto pregnanes. com \ - +2 Amfil+zv AHM-+zv . mmm mmm «mo Aomifim-+zv mom com . cow Aamlomxml+zv . S . com m&\ com OOH l Hma mma Figure 20. 104 Mass spectrum of the M0-TMS derivative of a pentahydroxy-pregnane-one that decreased in the urine of P885 exposed female rats. The molecular ion (m/z=771) and M+-31 (m/z=740) are observed, as are characteristic losses of M+-90-31, M+-2X90-31 and M+-3X90-31. com - z Aomit): AHMloml+zv + HNN AHMl+zv osm me omw ooo com AQMlooxml+zv AHMlomXMl+zv Ham A om: co: com com OOH mx Hmfi mNH 106 The lower end of the mass spectrum contains an intense frag- ment at m/z=188. This is a characteristic ion for a C-21 hydroxyl, C-20 methoxime configuration or a C-16 (or 15) hydroxyl, C-20 methoxime configuration with the C-21 posi- tion reduced. This second configuration can occur by reduc- t al., tion of the C-21 hydroxyl by gut microbes (Eriksson 1969). This particular steroid was observed to increase in the urine of PBBs-exposed subjects. This effect is most likely due to increased 63,7a,ISo(or 16a)-hydroxylation of corticosterone in the PBBs-exposed subjects. For example, likely structures for this steroid are 36,113,15a(or 160), 21-tetrahydroxy-56-pregnane-20-one or 3a,63,113,15a(or 16a)- tetrahydroxy-Sa-pregnane-ZO-one. The third steroid listed in Table 4 is also a tetrahydroxy-pregnene-one with a similar mass spectrum to peak 1 and may represent the 33-isomer of the first steroid. This steroid also increased in the urine of PBBs exposed subjects. The mass spectrum for peak 2 is shown in Figure 19. The high end of the mass spectrum shows the same characteristic ions (M+, M+-31, M+-31-90, etc.). The low end shows an intense fragment ion at m/z=170, which is a characteristic fragment of MO-TMS derivatives of C-15 hydroxy, C-20 keto steroids. Ions at m/z=129 and 191 also are characteristic fragments. The ion at m/z=129 is derived from an A-ring with either a 2,3-diTMS or a 5-ene-3-TMS configuration. The ion at m/z=191 is characteristic of polyhydroxylated steroids with TMS- hydroxyls on adjacent carbons. For example, a 2,3-diTMS 107 configuration in the A-ring will give a m/z=191 fragment. This steroid was decreased in the urine of the PBBs-exposed subjects. PBBs have been shown to inhibit So-reduction of testosterone tg li££2 (Newton gt _1., 1980, 1982). Thus, inhibition of So-reduction of corticosterone is one plausible mechanism by which PBBs could decrease the excretion of a urinary metabolite of corticosterone. Figure 20 shows the mass spectrum of a pentahydroxy- pregnane-one that was decreased in the urine of PBBs-exposed subjects. Characteristic losses of 31, 90+31, 2X90+31, 3X90+31 from the molecular ion are seen. The low end of the mass spectrum shows ions at m/z=191 and 129. As stated above, m/z=129 is derived from the A-ring (2,3-diTMS or a 5-ene-3-TMS configuration) and 191 is characteristic of polyhydroxylated steroids with TMS-hydroxyls on adjacent carbons. Reduction of this steroid in the urine of P885- exposed rats could have occurred by the same mechanism that resulted in a decreased excretion of the second steroid listed in Table 4 (i.e., inhibition of 5a-reduction). Because of the complicating aspects of the estrus cycle on urinary steroid excretion in female rats, it was decided at this point to use male rats exclusively as the experimental model. Attempts were also made to simplify the chromatogram by fractionating the hydrolyzed (free) steroid milieu with Sephadex LH-20 column chromatography by the method of Shackelton and Whitney (1980). The first experi- ment involved making multiple cuts (9 total) of the total 108 250 ml of eluant (data not shown). This resulted in greatly simplified capillary column gas chromatograms, but with extensive overlay of the elution of most components of the mixture. A second experiment was performed in which frac- tions were collected from 0-37 ml, 37-67 ml and 67-150 ml and analyzed by capillary column GC. These data are diSplayed in Figure 21. The top trace shows the chromato- gram obtained for the 0-37 ml fraction, the middle trace the 37-67 ml fraction, and the bottom trace the 67-150 ml fraction. Since all three traces seen in Figure 21 are relatively uncomplicated (i.e., when compared to the non-fractionated GC trace), and there was still overlapping of a large number of peaks, the experiment was repeated collecting fractions from 0-55 and 55-150 ml. These data are displayed in Figure 22. The top GC trace is for the 0-55 ml fraction and the bottom trace is for the 55-150 ml fraction. These chromatograms were still far simpler than chromatograms from non-fractionated urine. However, since there was still some degree of overlap, most apparent in the middle region of the chromatograms, it was decided to use the LH-20 fractions to obtain "clean" mass spectra for the steroids occurring in male rat urine and not for quantitative purposes at this time. Figure 21. 109 Capillary column GC traces obtained for three different fractions from Sephadex LH-20 column chromatography. The method of Shackelton and Whitney (1980) was used to chromatograph the free steroid fraction.' The top trace shows the chromatogram obtained with the 0-37 ml fraction, the middle trace is for the 37-67 ml fraction, and the bottom trace is for the 67-150 ml fraction. Chromatograms were obtained using a 60 m 08-1 fused silica capillary column (0.242 mm I.D., 0.1 um film thickness). .21.. n} .-. 3.1. 0-37 ml CA.!' F C— Cr ar.ff .... .r 1 ill) .llll 2 ..r ’0 C' 1“ 8' al.,“. 5. . a: co Ill]!!! " 1“ ‘6 . Ill 01),)vw . 3\. 0. ”ll llhlIllIll'lllli K\ V. G:- \' .bp. ‘1. I" w ‘ l .... N u... .... .f.(0 ..1..l{ “.1 ll. 5...... Hi 1 iii lllllllllll 7‘ 1‘ 67-150 ml C. If «...-r a... 1- (x «r a. I. as .... ill or 3.. U). l. l i ll- --llllll ill 3.11) 1) ‘1 l 5 .r Clitlllll . ll. Figure 22. 111 Capillary column GC traces obtained for two different fractions from Sephadex LH-20 column chromatography. The method of Shackelton and Whitney (1980) was used to chromatograph the free steroid fraction. The top trace shows the chromatogram obtained with the 0-55 ml fraction, and the bottom trace is for the 55-150 ml fraction. Chromatograms were obtained using a 60 m DB-I fused silica capillary column (0.242 mm 1.0., 0.1 um film thickness). 2. c “'V ‘»‘v ‘fl‘f 0-55 ml Cc‘a‘! m .10 I", 1'.” 51.12” .; ll -l'L‘.‘ - ‘ab'LV E ¥%‘|H m 4"l‘, :v' I (9'03 w 85 {6'45 til-VS Lh'yi verbs. UB‘S': CU'hb 5"". 75's". 64 cu ‘ . cc'ib ’ “a L ; Ll'|b PV'V' 1.8". La ,-,,, ...“... 6‘ '6' C (.2. 7.? 113 In the following experiment, the Dent gt gt. (1976) protocol was used with male rats (150 mg/kg PBBs adminis- tered to 21-day-old rats). Urines were collected at 28 days (pre-puberal) and 60 days (post-puberal). The concentration of total steroids in the pre-puberal samples was not suffi- cient for accurate mass spectral analysis, especially in the corticosteroid region where differences were expected to occur based on the previous results. Since approximately 4 to 5 times as much urine was collected from the post-puberal animals, these samples were chosen for detailed character- ization. Samples were prepared by Method II (see Section II) and analyzed by both capillary GC and GC/MS/DS. As with the previous experiment, most peaks were not quantitatively different between the control and P885 groups, and no obvious qualitative differences were noted. Therefore, only data for those peaks where mass spectral interpretation was informative are presented below (Table 5). Mass spectral analysis (see below) characterized the top three steroids listed in Table 5 as tetrahydroxy-pregnane-ones and the bottom four steroids as Sa-pregnane-3,11,15,20,21-pentols. Of these compounds, two of the tetrahydroxy-pregnane-ones increased statistically (t-test, (0.05) and one was unchanged, while two of the four pregane pentols decreased. Mass Spectral characteristics of the tetrahydroxy- pregnane-ones are similar to what has previously been described, with M+, M+-31, M+-31-90, M+-31-2X9O and M+-31-3X9O 114 Table 5. Urinary metabolites of corticosterone in control and PBBs exposed adult male rats. Amount/mg creatinine1 Average (510.) Control PBBs R. I. 774')" THE-‘4') Tetrahydroxy-pregnane-ones 3015 5.08 (1.65) 5.02 (2.62) 3106 0.20 (0.06) 1.19 (0.19)* 3164 1.70 (0.29) 16.0 (0.95)* 5-Pregnane-3,11,16,20,21-pentols 3251 1.08 (0.41) 1.70 (0.62) 3270 3.17 (1.18) 0.84 (0.65)* 3307 4.18 (0.65) 6.46 (3.61) 3306 1.75 (0.66) 0.44 (0.04)* 1 Values are average of four samples and are normalized to the internal standard and urinary creatinine. Standard deviation is in parentheses. * Significantly different (p<.05). 115 occurring in the high end of the mass spectrum. Although mass spectral interpretations of the peaks at retention index 3015 and 3106 established that these compounds were tetrahydroxy-pregnane-one metabolites of corticosterone, the sterochemistry could not be determined. However, the corticosterone metabolite at 3164 had a very prominant ion at m/z = 188, a characteristic fragment for a C-21, TMS- hydroxyl, C-20 methoxime configuration or a C-16 (or 15) TMS-hydroxyl, C-20 methoxime configuration with the C-21 position reduced. Likely structures for this steroid are 3a,113,15a(or 15a), 21-tetrahydroxy-So-pregnane-ZO-one or 3o,63,113,156(0r 16a)-tetrahydroxy-5o-pregnane-20-one. The bottom four steroids in Table 5 are 56-pregnane- 3,11,16,20,21-pentols. These steroids have previously been reported to occur in the urine of conventional male rats (see Introduction, Section III). The mass spectrum of a 5a-pregnane-3,11,16,20,21-pent0l is shown in Figure 23. Characteristic ions at m/z = 625 (MT-103), m/z = 535 (MT-103-90), m/z = 445 (MT-103-2X90), m/z = 355 (M+-103-3X90) and m/z = 265 (M+-103-4X90) occur for this series of steroid stereoisomers. The absolute configura- tions of the steroids listed in Table 5 could not be deter- mined. However, the order of elution of the a- and 3-enatiomers of 3,16 and 20-hydroxy-substituted steroids indicates that the two steroids which decreased in the P885 subjects must have the same configuration in the 20 position, but different configurations at the 16 position. Figure 23. 116 Mass spectrum of a 56-pregnane-3,11, 16,20,21-pentol. Characteristic ions at m/z = 625 (MT-103), m/z = 535 (M+-103-90), m/z = 445 (M+-103-2X90), m/z = 355 (M+-103-3x90) and m/z = 265 (MT-103-4X90) occur for this series of steroid epimers and enatiomers. The fragment at m/z = 191 is very characteristic of polyhydroxylated steroids with TMS-hydroxyls on adjacent carbons. v 265 129 283 44w 235 21 173 191 159 r"‘""'|"""" 150 I 3 lie-(I'JWJAL Iii cm » . -. fl 1 I ‘ T ' I r . 285 MW M ‘ n. ' b- o. c. '2' Li H ‘3 Li cit-I 11:1 ' ”‘0 240 E.“- a. O h '-' 18 2:20 2 - .0 h .121 200 ‘- Q 188 1 - L1 t.‘ - 1:1 14 “.20 a Q 0 ~' I . .' K c h- 117 445 - p b p p - v ; In D 9-0 h D b >8 - D ‘0 625 535 jI'ITT'I 36.8 r271: 1 a -- LI 1.17.30 I .7 30 84 «ii ..8 -- —- 1 El £113 E. E . “LN-:1 . I" - '_ 91:1 :53] 4 Figure 24. 118 Capillary GC trace of urinary steroids obtained for a group of four male rats before (control) and after ("germ-free") antibiotic treatment. Trace was obtained using a 60 m DB-l fused silica capillary column. MO-TMS derivatives of urinary steroids were prepared using Method II (Section II). Arrows show where peaks have been attenuated or are absent in the "germ-free“ urine. I”? :3 1:15’55 31 '55 ué'vs :z‘vs foe-‘L‘S e ("z-:55 I' .II L c-S {S —’=1 .1; 5 9858);? :5 :11: ”1:15 ‘24” fish bv'bt’ S 321:1 ‘ev 59°15? ch4 -.-.Lo CONTROL "GERM-FREE" 120 o-Stereoisomers elute before 3-stereoisomers at these posi- tions (Eriksson, 1971b). Also, the four possible 36-isomers will elute before any of the 33-isomers (Eriksson, 1971b). The order of elution for the 16 and 20 position isomers is 166,ZOa-, 15a,208-, 163,206- and 163,203-. Therefore, the two pregnane pentols that decreased in the P885 group must differ at the 16 position and must have the same steroichem- istry at the 20 position. Of course, these steroids could either be 36- or 33-hydroxy-pregnanes. Attempts were made to simplify the urinary steroid metabolic profile by making the subjects germ-free. This was atempted using a published method (Rowland gt gl., 1980) and involved addition of bacitracin, neomycin sulfate and streptomycin sulfate at a concentration of 2 mg/ml to the drinking water. Figure 24 shows the capillary GC trace of urinary steroids obtained for a group of four male rats before antibiotic treatment (control) and after antibiotic treatment ("germ-free"). A number of peaks were either absent or greatly attenuated in the "germ-free" chromato- grams (none of these peaks could be identified by GC/MS). However, there are two problems with this procedure. First, the subjects would not drink the antibiotic contaminated water until they were very thirsty (2-3 days) and then only sparingly. Second, the animals were not completely germ-free since bacterial cultures of fecal material from all but a few of the subjects were positive. DISCUSSION A number of steroids reported to occur in male and female rat urine were not detected by the methods employed in these studies. These steroids include several C-19, 0-2,3 metabolites of testosterone and C-21, 0-2,3,4 metabolites of corticosterone and progesterone. It is possible that strain differences or differences in the intestinal flora could influence the urinary steroid metabolic profile. However, the most likely explanation for our inability to detect many of the steroids reported to be excreted in the urine of male and female rats is related to the initial volume of urine. The earlier studies were all qualitative in nature and large volumes of urine were used (100 ml or more for each sample preparation). In some of the GC/MS analyses in the present study, there were occurrences of weak ion currents that suggested the presence of some of these compounds. For example, in a few analyses, the presence of weak ion currents at m/z = 436 (MT), 421 (MT-15), 406 (MT-30) and 326 (MT-90) suggested the presence of a TMS derivative of an androstanediol. There was also some indication of the presence of androstanetetrol- TMS compounds (M+=612). Larger volumes of urine need to be pooled in order to detect and identify these compounds. 121 122 Although certain differences were noted between the P885 exposed and the control subjects in the urinary concentra- tions of some metabolites of corticosterone, none of these steroids could be fully characterized. Thus, it was impos- sible to describe a specific effect of P885 exposure as the result of an increased or decreased activity of a specific metabolic step (i.e., increased 63-,7o,15a or 16a- hydroxyl- ation of corticosterone for example). The large number of non-steroid compounds and the rela- tively low concentrations of steroids (i.e., compared to humans) in rat urine make studies of this type difficult and time consuming. The extensive enterohepatic circulation and metabolism of steroid hormones by gut microbes in the rat further complicated the interpretation of steroid profiles in this species. In summary, PBBs administration was noted to alter the urinary steroid metabolic profile in male and female rats. Due to the complexity of the rat urinary steroid profile, the presence of a large number of non-steroidal substances, the extensive enterohepatic circulation of steroids and metabolism by gut microbes and the lack of reference com- pounds, it was impossible to accurately characterize the exact nature of this phenomenon. However, it is most likely that at least some of the increased urinary excretion of steroids seen in the P885 group is related to stimulation of steroid hydroxylases. It is also likely that decreased urinary excretion of steroids in the P885 group is related to inhibition of steroid reductases. SECTION IV Human Studies CHAPTER IV INTRODUCTION This section describes the experiments using human subjects. The first series of experiments were designed to determine daily variations in the excreted concentrations of urinary steroids using "spot“ urine collections and normalizing to urinary creatinine concentration. Also determined were daily variations in the excreted concentrations of urinary steroids using 24 hour urine collections and normalizing to total amount per 24 hours. It was necessary to determine the relative variability of using a “spot“ urine collection protocol compared to that obtained for a 24 hour urine collection protocol to ascertain if "spot" urine collections were suitable for subsequent experiments. After establishment of the expected variability in normals, the urinary steroid metabolic profiles of a group of humans accidentally exposed to polybrominated biphenyls were compared to the steroid profiles obtained for an appropriate control group. Catabolism of steroid hormones in man. The following overview of some aspects of steroid metabolism in man is presented as background for the discussion of results on human steroid profiles to follow. Because of the complexity 123 124 of steroid hormone metabolism and urinary excretion, it is necessary to present this material for the reader to understand and appreciate these results. More detailed accounts of this subject have been presented by Makin (1975) and Schulster gt g1. (1976). Although the urine contains a surprisingly large number of steroid metabolites, these compounds are formed by rela- tively few enzymatic conversions. One of the common struc- tural features of physiologically active steroid hormones, with the notable exception of the estrogens, is a 4-ene-3- one A-ring configuration. Reduction of this configuration will generally result in a loss of biological activity although there are exceptions to this generalization (i.e., Sa-reduction of testosterone enhances its activity in several target tissues). Reduction of the 4-ene-3-one system to a 3-hydroxy-5-dihydro configuration results in one of four stereoisomers: 36-01-53; 33-01-53; 36-01-56; 33-01-5o. While all of these transformations do occur, the relative activities of the liver enzymes involved varies from species to species and also with sex. In lower animals the 33-hydroxy configuration predominates while in man 36-hydroxy steroids predominate. In man, the first step in reduction of the A ring involves a 4-ene-53-reductase or a 4-ene-56-reductase and is an NADH or NADPH requiring reaction. In the second step either a 36-hydroxy steroid dehydrogenase or a 33-hydroxy- steroid dehydrogenase acts on the partially reduced steroid. 125 The 4-ene-56-reductase is found in the microsomal fraction of the liver, adrenal, testes and ovaries while the 4-ene- 53-reductase is found in the soluble fraction of the same tissues. The 36-hydroxysteroid dehydrogenase is found in the soluble and microsomal fractions of the liver and kidney and the microsomal fraction of the testes, ovaries and adrenal, while the 33-hydroxysteroid dehydrogenase is found in the microsomal fraction of the liver, testes, ovaries and adrenal. A second general pathway for reductive inactivation of steroids involves reduction of the 20-ket0 functional group of C-21 steroids to a 20o- or a 203-hydroxy group. Both enzymes involved in these reactions, 206- and 203-hydroxy- steroid dehydrogenase, require NADPH or NADH. The 200- hydroxysteroid dehydrogenase is found in the soluble frac- tion of liver, kidney, muscle, adrenal, testes, ovaries and placenta, whereas the 203-hydroxysteroid dehydrogenase is found in the soluble fraction of liver, muscle, testes and ovaries. A third general pathway for inactivation of steroid hormones occurs through formation of a 17-keto derivative of 17-hydroxy C-19 steroids or 17-hydroxy C-21 steroids, the latter involving the cleavage of the C-20,21 side chain. For example, a 173-hydroxysteroid dehydrogenase converts testosterone to 4-androstene-3,17-dione which can then undergo A-ring reduction to form either androsterone (36- hydroxy-So-androstane-l7-one) or etiocholanolone (3a-hydroxy-53-androstane-l7-one). 126 Catabolism of the C-19 steroid hormones. The most impor- tant androgen produced by the testes is testosterone. Testosterone is derived from androstenedione, which also has weak androgenic activity. Another important source of androgens is the adrenal cortex where large quantities of dehydroepiandrosterone (DHEA) are produced. Androstenedione and 113-hydroxyandrostenedione are also produced in the adrenal cortex. As was previously mentioned, one pathway for the catabolism of testosterone involves conversion first to androstenedione with subsequent reduction of the A-ring forming either androsterone or etiocholanolone. A second pathway involves the reduction of the A-ring of testosterone to yield either So-androstanediol (So-androstane-36,173- diol) or 53-androstanediol, both of which can be excreted as the glucuronide conjugate. DHEA can be hydroxylated at either the 7 or 16 positions (both o-hydroxylations) or the 17-keto group can be reduced to a 173-hydroxy. These metabolites are conjugated with glucuronic acid before excretion in the urine. Catabolism of progesterone. Progesterone and 17a-hydroxyprogesterone primarily undergo reduction to 36-hydroxy-53-pregnanes. Progesterone undergoes A-ring reduction to yield pregnanolone (3a-hydroxy-53-pregnane-20- one) and subsequent reduction of the 20-keto group to a ZOo-hydroxy yields pregnanediol, the major metabolite of progesterone. Both the pregnanediol and pregnanolone are 127 excreted as glucuronides. 17o-Hydroxyprogesterone undergoes A-ring reduction to yield 36,17o-dihydroxy-53-pregnane-20- one which can be reduced to pregnanetriol (SB-pregnane- 36,17o,206-triol). Both of these metabolites are excreted as glucuronides. 17o-Hydroxyprogesterone can also undergo A-ring reduction and cleavage of the C-17,20 bond to yield etiocholanolone. Catabolism of the adrenal steroids. Aldosterone also undergoes A-ring reduction with the major metabolite being 36,53-tetrahydroaldosterone which occurs in urine as the glucuronide conjugate. Other metabolites occur but only in small amounts. Glucocorticoids also undergo A-ring reduc- tion. In man the major glucocorticoids secreted by the adrenal are cortisol and cortisone while corticosterone predominates in rats, rabbits and mice. The 11-deoxy derivatives of cortisol and corticosterone are also produced by the adrenal. Both the 4-ene-5o-reductase (microsomal fraction) and the 4-ene-53-reductase (soluble fraction) will reduce these steroids. Cortisol is primarily converted to the 53-isomer and corticosterone to the 56-isomer. Further reductions occur at the 3, 11 and 20 positions. Cortisol and cortisone are interconvertible by means of an 113-hydroxysteroid dehydrogenase (microsomal fraction of liver and adrenal). The principal metabolites of cortisone are 30,170,21' trihydroxy-53-pregnane-11,ZO-dione (tetrahydro E; THE) and the ZOa-hydroxy or 203-hydroxy derivatives of THE 128 (o-cortolone and 3-cortolone). The 56-derivatives of these metabolites are also formed but to a lesser extent. Cortis- ol is also primarily converted to a 53-derivative and its major metabolites are 36,113,17o,21-tetrahydroxy-53- pregnane—ZO-one (tetrahydro F; THF) and the ZOa-hydroxy and 203-hydroxy derivatives of THF (a-cortol and 3-cortol). The major metabolite of corticosterone is its 36-hydroxy-56- reduced derivative (36,113,21-trihydroxy-56-pregnane-20-one; aTHB) although some of the 53-derivative (THB) is produced. Both metabolites are excreted in the urine as the glucuro- nide conjugate. Some free cortisol and corticosterone are also usually present in the urine. Catabolism of the C-18 steroids. The most physiological- ly active estrogen is estradiol-173 which is interconverti- ble with the somewhat less active estrone via action of a 173-hydroxysteroid dehydrogenase. Estrogens can undergo hydroxylation at C-2,4,6,15,16 and 18 and many different metabolites appear in the urine. However, the quantitative- ly most important estrogen metabolite is estriol (1,3,5[10]- estratriene-3,16a,173-triol). Estriol appears in the urine as a glucuronide. Estrone sulfate is also seen in the urine, but during pregnancy its major conjugate is the glucuronide. Formation of steroid conjugates. The enzyme responsible for formation of steroid sulfates is a sulfokinase requiring ATP and Mg2+ which is found in the soluble fraction of the liver, testes, adrenal zona fasciculata and reticularis. 129 Glucuronides are formed by means of a glucuronyl transferase found in liver microsomes. This reaction requires UDP- glucuronic acid. EXPERIMENTAL Materials. All materials were obtained from the sources previously described in Section II. Isolation of steroids. Steroids were isolated by Method 11 described in Section II. Gas chromatography. Urinary steroid metabolic profiles were generated using a 60 m DB-1 bonded phase fused silica capillary column as previously described in Section II. Gas chromatography/mass spectrography. Urinary steroid metabolic profiles were generated by GC/MS/DS as previously described in Section 11. Collection of urine. In the first series of experiments 24-hour urine samples were collected for six days, starting after the first void morning urine and ending with the first void morning urine the following day. Spot urine samples consisted of the first void morning urine. For the PBBs studies, spot urine samples were taken at various times of the day. Selection of subjects for PBBs study. Samples were obtained by the Michigan Department of Public Health from patients enrolled in the "Long-Term PBB Study". A randomly selected high PBB subgroup (PBB t 50 ppb) and an age- and sex-matched low PBBs subgroup (PBBs 5.2 ppb) were compared. A complete medical history was taken for all individuals enrolled in the study at the time of urine collection (a copy of the qugstionnaire used is displayed in Appendix I). 130 131 For the present study these questionnaires were used to select the most appropriate samples from those made available. Appendix II lists information for those factors deemed most important in sample selection for individuals used in the present study. Age, diabetes, kidney disease, liver disease, smoking and medications were the most important consideration. Individuals with diabetes, kidney disease, liver disease, or individuals who smoked were excluded from the study. Individuals over the age of 70 were excluded, as were individuals on medications known to affect microsomal mixed function oxidase activity (i.e., anticonvulsants). The ages of the low PBBs group were 21,21,33,33,39,42,47,48,49,50,54,60 and 69. The ages of the high PBBs group were 20,21,23,28,36,42,48,52,58,58,64,65 and 65. Other medical factors (such as recent surgery, high blood pressure) were also taken into consideration. Because of the complicating aspects of the menstrual cycle on excretion of urinary steroids in females, it was decided that male subjects would be used exclusively in the present study. 132 RESULTS The first phase of the human experiments involved quali- tative and quantitative determination of daily variations in the excreted levels of urinary steroids when normalized to 24-hour excretion rates and when normalized to urinary creatinine concentration. This was necessary to ascertain the relative variability for each mode of normalization. Although 24-hour urine collections have been described by other investigators as being more reproducible than spot collection, they have one major disadvantage in that one is never sure that the total urine output for the entire 24- hour period was collected unless the collections are strict- ly supervised. It was decided that if the daily variation seen when data are normalized to urinary creatinine concen- tration is similar to the variation seen when normalizing to total 24-hour excretion rates, then this mode of normaliza- tion would be preferable in the current studies. The 24-hour urinary steroid excretion pattern of a normal adult male over a 6 day period is shown in Table 6. Values were calculated by the MSSMET program in the follow- ing manner. The integrated area of the profiles of 'desig- nate' ions (ions chosen for quantification) were divided by the integrated area of the designate ion of the internal 133 Table 6. Urinary steroid metabolic profiles of a normal adult male: 24 hour excretion patterns over a 6 day periodl. Amount/24 hours2 Retention Day Name Index 1 2 3 4 5 6 Androsterone 2532 2660 2420 2770 3310 2130 1780 Etioiholanolone 2547 2530 2420 2610 3770 2340 1820 DHEA 2597 484 381 623 1890 1580 1010 ll-keto-Ez 2634 225 188 240 266 210 170 113-hydroxy-A 2715 786 684 587 563 688 659 113-hydroxy-E2 2731 448 347 444 437 309 334 166-hydroxy-DHEA 2774 1780 1890 1520 2610 1450 975 Pregnanediol 2784 1930 1890 2060 2710 1820 1210 Pregnanetriol 2815 3570 3100 3320 3130 2190 2130 THS 2845 -- -- 15.7 40 -- -- THE2 2978 2060 1510 1610 1680 1330 1600 THA2 2979 31.7 93.8 93.3 -- 101 101 THB 3000 154 23.9 114 157 96.7 100 allo-THBZ 3016 269 29.6 240 186 101 61.8 all -THE 3030 182 109 139 149 68.6 88.3 THF 3030 1130 982 1000 1100 682 940 allo-THF 3040 698 456 554 486 360 428 Cortolonez 3059 485 451 461 459 299 437 B-Cortol 3088 428 376 271 268 158 294 3-Cortglone2 3090 759 573 454 357 274 486 Cortol 3130 220 196 197 195 136 182 1 Urine was collected starting after the first void morning urine and ended with the first void morning the following day. 2 Values were calculated by the MSSMET program as follows: the integrated area of the GC peak of the designate ion for each steroid was divided by the integrated area of the GC peak of the designate ion for the internal standard. This ratio was divided by the number of ml of urine used, multiplied by the number of 39 of internal standard added, and multiplied by the total volume of urine collected over the 24 hour period (ml). This value was converted to actual ug of sample for those steroids where a relative resBonse factor had been determined (designated by a superscript ). 3 Abbreviations: Androsterone, 36-hydroxy-So-androstane-17-one; Etiocholanolone, 36-hydroxy-53-androstane-17-one; DHEA, dehydroepian- drosterone, 33-hydroxy-5-androstene-17-one; 11-keto-E,11-keto- etiocholanolone, 36-hydroxy-53-androstane-11,17-dione; 113-hydroxy-A, 1I3-hydroxy-androsterone, 36,113-dihydroxy-Sa-androstane-17-one; 113- hydroxy-E, 113-hydroxy-etiocholanolone, 36,113-dihydroxy-53-andro- stane-17-one; 166-hydroxy-DHEA, 33,16a-dihydroxy-S-androstene-l7-one; 134 Table 6 (continued) pregnanediol, 53-pregnane-3a,ZOo-diol; pregnanetriol, 53-pregnane- 36,176,206-tri0l; THS, 36,176,21-trihydroxy-53-pregnane-20-one; THE, 3a,17a,21-trihydroxy-53-pregnane-11,20-dione; THA, 36,21-dihydroxy- 53-pregnane-11,20-dione; THB, 36,113,21-trihydroxy-53-pregnane-20- one; allo—THB, 36,113,21-trihydroxy-Sa-pregnane-ZO-one; THF, 30,118, 17a,21-tetrahydroxy-53-pregnane-20-one; allo-THE, 36,176,21- trihydroxy-So-pregnane-ll,20-dione; allo-THF, 3o,113,17a,21-tetra- hydroxy-53-pregnane-11-one; cortolone, 36,17o,20a,21-tetrahydroxy-53- pregnane-ll-one; 3-cortolone, 36,176,203,21-tetrahydroxy-53-pregnane- 11-one; 3-cortol, 53-pregnane-3a,113,17o,203,21-pentol; cortol, 53- pregnane-36,113,17a,206,21-pentol. 135 standard. These ratios were divided by the volume (ml) of urine used (20), multiplied by the amount of internal stan- dard added (10 39), and multiplied by the total volume of urine collected over the 24 hour period (in ml). These relative concentrations were converted to actual ug of steroid per 24 hour whenever relative response factors had been determined. The morning spot urinary steroid excretion patterns of a normal adult male over a 6 day period are shown in Table 7 (not the same subject as in Table 6). Values were calculat- ed by the MSSMET program in the following manner. The ratios of integrated areas of the designate ions to the integrated area of the designate ion of the internal stan- dard were divided by the volume (ml) of urine used (20) and the concentration of creatinine (mg/ml) and multiplied by the amount of internal standard added (10,000 ng). These values were converted to actual ng of sample per ng creatinine whenever relative response factors had been determined. Excretion patterns of steroids were noted to be fairly consistent from day to day for major components (i.e., androsterone, etiocholanolone, pregnanediol, pregnanetriol, THE, THF, the cortolones and cortols). The large variation in the excretion pattern of DHEA was expected, based upon work by others (Van de Calseyde gt gl., 1972) which indicat- ed that DHEA excretion is related to stress. Variations in the excretion patterns of some of the minor metabolites of adrenal corticosteroids were probably due to difficulties 136 Table 7. Urinary steroid metabolic profiles of a normal adglt male: Morning spot urine excretion patterns over a 6 day period. Amount/mg Creatininez Day Name3 1 2 3 4 54 6 Androsterone 1460 2050 1950 1410 838 1832 Etiogholanolone 1020 1340 1340 953 456 1240 DHEA 950 250 830 1080 180 643 11-keto-E2 133 117 152 148 54.1 190 113-hydroxy-A 519 359 637 452 256 573 113-hydroxy-Ez 86 100 81.9 91.6 33.9 59.8 166-hydroxy-DHEA 104 125 179 215 83.4 _- Pregnanediol 1340 1420 1460 819 268 745 Pregnanetriol 958 1370 1010 931 381 1090 THS 19.2 31.3 12.5 16.1 7.32 58.8 THE2 545 503 787 528 303 626 THA2 30.9 48.3 90.5 59.7 47.7 210 78H2 43.8 44.8 79.6 47.9 36.1 58.6 alio-THB2 130 69.9 154 72.5 74.9 112 all -THE 40.4 46.0 67.2 39.1 20.6 57.0 THF 335 227 406 285 168 357 allo-THF 204 193 237 172 127 209 Cortolonez 123 143 202 140 79.3 165 B-Cortol 46.0 47.2 60.3 45.1 32.4 38.3 3-Cortglone2 114 143 169 126 81.9 130 Cortol 57.6 50.3 67.5 42.9 34.1 47.5 1 Urine was collected with the first void morning urine. 2 Values were calculated by the MSSMET program as follows: the integrated area of the GC peak of the designate ion for each steroid was divided by the integrated area of the 60 peak of the designate ion for the internal standard. This ratio was divided by the number of ml of urine used and the concentration of creatinine (mg/ml) and multiplied by the amount of internal standard added (10,000 ng). This value was converted to actual ng of sample for those steroids where a relative reponse factor had been determined (designated by a superscript ) 3 Abbreviations are defined in Table 6. 4 Values are consistently low and thus supect. 137 in detecting designate ions of these substances, and by the sharp gas chromatographic peaks of those that were present. As discussed in Section II, the capillary column was delib- erately overloaded somewhat, such that larger peaks exhibit- ed a degree of 'fronting' with a slight overlapping of iso- mers, to detect and quantify these minor metabolites. These analyses represented a reasonable balance of the need for good gas chromatographic separation with the sample size required for adequate quantification by MSSMET. Another problem which may contribute to errors in the quantification of minor metabolites arose when contributions to designate ion currents resulted from much larger co-eluting or adja- cent peaks. This problem could be avoided by selection of ions that were unique, whenever possible. For example, intense ions in the THA mass spectrum all have a contribu- tion from the much larger THE peak, except m/z 431, which is unique to THA and therefore the best ion for quantification of THA. Table 8 shows the average urinary steroid excretion pat- terns over a 6-day period in absolute amounts for a 24 hr collection and relative to creatinine for a spot urine col- lection. Also shown are the designate ions used for quanti- fication and the calculated relative response factors. Although the values listed in Tables 6 and 7 for those steroids for which a relative response factor was not avail- able are not in absolute amounts, most of these values are within a factor of 2 or 3 of the actual amount since 138 Table 8. Average urinary excretion patterns over a 6 day period in absolute amounts for a 24 hour urine collection and relative to creatinine for a morning spot urine collection1. 24 Hour Morning Spot Designate Relative Collection Sample Name2 ion m/z Response4 ug/24 hours ng/mg creatinine DHEA 260 1.47 950 1 680 660 1 410 ll-keto-E 300 1.30 220 1 38 130 1 50 113-hydroxy-E 448 0.98 390 1 7O 75 1 27 THS 474 2.66 - - 24 1 21 THE 488 1.13 1600 1 270 550 1 170 THA 431 1.91 70 1 48 81 1 73 THB 474 1.08 110 1 53 52 1 17 allo-THB 474 1.13 140 1 110 100 1 39 THF 472 1.67 970 1 180 310 1 9O Cortolone 449 0.24 430 1 73 140 1 45 3-Cortolone 449 0.45 480 1 190 130 1 29 Cortol 343 0.46 190 1 31 50 1 13 1 Values are the average 1 standard deviation over a 6 day collection period. Values for the 24 hour urine collection were calculated as described in Table 6 and values for the morning spot urine collection were calculated as described in Table 7. 2 Abbreviations are defined in Table 6. 3 Ion used in quantitation (designate ion). See Sections I and II for more detail. 4 See Section II for calculation of relative response factor. Values were determined using an LKB-2091 GC/MS. 139 relative response factor values usually fall in the range of O.33-3.0. Assuming that the relative response factors for these steroids are in fact within this range, the values in Tables 6 and 7 fell well within the range of reported urinary steroid excretion patterns for adult males (i.e. 17-keto-steroids, 7-20 mg day'1; 17-hydroxy-steroids, 2-12 mg day'1; pregnanetriol, 0.5-3.0 mg day‘1). The one exception was the apparently high excretion rate of pregnanediol assumed from the relative value in Tables 6 and 7. These values are very different from absolute levels because the designate ion (m/z 117) of the trimethylsilyl derivative of pregnanediol is a particularly major propor- tion of total ionization and the relative response factor is therefore unusually low. Previous investigations have demonstrated large inter- individual variations in the excretion rates of urinary steroids (Pfaffenberger and Horning, 1977; Jurigskay and Kecskes, 1978; Moyer gt gt., 1978; Van de Calseyde gt gl., 1972; Fantl and Gray, 1977; Bailey gt gl., 1974; Vbllmin, 1971; Setchell gt gl., 1976). Of course, certain variations in excretion in females are related to pregnancy and the estrus cycle, but even with males, a wide range of values have been reported. For example, some of the average values that have appeared in the literature for 24 hour excretion rates of androsterone in normal adult males (in mg per 24 hour) are: 2.810.4, n=12 (Jurigskay and Kescskés, 1978); 2.215.0 (12 SD), n=23 (Moyer gt gl., 1978); 3.811.1, n=15 140 (Van de Calseyde gt gl., 1972); 2.7 with a range of 1.9-4.2, n=8 (Fantl and Gray, 1977); 2.03 with a range of 1.06-3.22, n=12 (Bailey gt_gl., 1974); and 2.5 with a range of 1.7-2.6, n=2 (Vbllmin, 1971). It is interesting that such a discrepancy exists in the literature for average excretion rates, a discrepancy that is apparently related to differences in methodology. Etiocholanolone also exhibits wide variations in excretion rates, with reported average values ranging from 1.2-4.8 mg per 24 hour (Jurigskay and Kecskes, 1978; Moyer gt gt,, 1978; Van de Calseyde gt gt., 1972; Fantl and Gray, 1977; Bailey gt gl., 1974; Vbllmin, 1971). DHEA exhibits the widest range in excretion rates (0.0-7.9 mg per 24 hour). Increases in the excretion of this steroid have been correlated with stressful situations (Spiteller, 1978). Pfaffenberger and Horning (1977) reported a minimum of 0.01, a maximum of 3.6 and an average of 0.7 mg DHEA/g creatinine in 21 male subjects. In the two individuals examined in the current study the urinary excretion rates of DHEA ranged from O.38-1.9 mg per 24 hour for the 24 hour collection to O.18-1.1 39 mg-1 creatinine for the morning spot collection. Values given in Tables 6-8 for the other 17- keto urinary steroids also agree well with the values reported by other investigators (Pfaffenberger and Horning, 1977; Jurigskay and Kecskes, 1978; Moyer gt gt., 1978; Van de Calseyde gt gt., 1972; Fantl and Gray, 1977; Bailey gt gl., 1974; VUllmin, 1971; Setchell gt l., 1976). 141 It has been reported that urinary excretion rates of the major metabolites of the corticosteroids also exhibit large inter-individual variations. Values ranging from 2.9-7.2 mg per 24 h and 1.1-8.1 mg/g creatinine have been reported for THE (Pfaffenberger and Horning, 1977; Van de Calseyde gt 31., 1972; Fantl and Gray, 1977; Vollmin, 1971). Values of .1-3.0 mg per 24 h and 0.44-2.9 mg/g creatinine have been reported for THF (Pfaffenberger and Horning, 1977; Fantl and Gray, 1977). Our values for excretion rates of the minor corticosteroid metabolites (i.e. THS, THA, THB, allo-THB, allo-THE) also agree with values reported by Setchell gt gt. (1976) and Pfaffenberger and Horning (1977). In summary, the day to day variation in the urinary excretion of steroids when calculated per mg creatinine was comparable to that seen when the excretion rate was expressed as amount per 24 hours and was within the limits arbitrarily set at the beginning of these investigations (IS-20% for the major urinary steroids). Also, the data for urinary steroid concentrations obtained by automated reverse library search of selected mass chromatograms were similar to those reported by other investigators using different methods of analysis. Effect of accidental expgsure to PBBS on urinary excretion of 63-hydroxycortisol. A number of drugs are reported to be inducers of microsomal mixed function oxidases in man as well as other species. These include such agents as phenobarbital, antipyrine, rifampicin, 142 phenytoin, and carbamazepine (Ohnhaus and Park, 1979; Roots gt gl., 1979). 63-Hydroxyc0rtisol, a polar metabolite of cortisol excreted in the urine unconjugated (Frantz gt gl., 1961; Thrasher gt El). 1969; Chamberlain, 1971; Werk gt gl., 1964; Yanaji gt _1., 1969; Berman and Green, 1971), has been reported to be a reliable indicator of hepatic microsomal MFO enzyme activity (Saenger gt gl., 1981; Berman and Green, 1971; Ohnhaus and Park, 1979; Stevenson gt gl., 1972; Poland t 1., 1970; Roots gt 11., 1979). Saenger g a_l. (1981) reported that 63-hydroxycortisol increased 4- to 7-fold in the urine of children on anticonvulsant therapy. The ratio of 63-hydroxycortisol to total urinary 17-hydroxycortisol and free cortisol also increased. Phenytoin and carbamaze- pine have been reported to increase urinary 63-hydroxy- cortisol (Roots gt gl., 1979) as have antipyrine, phenobar- bital and rifampicin (Ohnhaus and Park, 1979). Other factors are also known to effect urinary excretion of 63- hydroxycortisol. This urinary metabolite of cortisol is relatively high in pregnancy (Frantz gt gl., 1960; Katz gt gt., 1962), in certain cancer patients (Werk gt gl., 1964) and in the newborn (Daniilescu-Goldinberg gt_gl., 1974). Estrogen therapy was reported to increase 63-hydroxycortisol excretion (Katz gt gl., 1962). Thyroid dysfunction has also been reported to increase 63-hydroxycortisol excretion rela- tive to total urinary 17-hydroxycorticosteroids (Yamaji gt gl., 1969). The ratio of 63-hydroxycortisol to 17-hydroxy- corticosteroids is reported to be slightly higher in females than males (Thrasher gt gl., 1969). 143 The ability of certain drugs to increase urinary excre- tion of 63-hydroxycortisol is classified as a "phenobar- bital-like" effect on hepatic microsomal MFO enzyme systems. This is an important consideration since one of the effects of certain PBBs congeners in laboratory animals is a "pheno- barbital-like" effect on hepatic MFO enzymes. In particul- ar, those congeners with 2 or more ortho bromines have a "phenobarbital-like" effect in that they induce liver micro- somal enzymes usually induced by phenobarbital and cause a proliferation of hepatic endoplasmic reticulum. Congeners without bromines in the ortho position induce microsomal enzymes usually associated with 3-methylcholanthrene expo- sure. Those congeners with one ortho bromine show moderate toxicity and are "mixed" inducers of liver microsomal enzymes (having both 3-methylcholanthrene and phenobarbital- like effects). Individual components of the commercial mixture of PBBs which contaminated Michigan all contained at least one ortho bromine and 89% of the total mixture contained two ortho bromines (Moore and Aust, 1978; Moore _t__l., 1978, 1980). It would be reasonable to expect that one of the possible effects of this particular mixture of PBBs would be a "phenobarbital-like" effect in the exposed human population. Therefore, it follows that the exposed human population might have increased levels of urinary 63-hydroxycortisol. The electron impact mass spectrum of the methoxime- trimethylsilyl derivative of 63-hydroxycortisol is given in 144 Figure 25. The molecular ion is seen as are characteristic losses of 15 (CH3+), 31 (OCH3+), 90 + 31 (TMSiOH+ + OCH3+), 2X90 + 31 and 3X90 + 31. This derivative had a retention index of 3313 on the 50 m DB-l capillary column and 3315 on the 3% OV-101 packed column. Table 9 shows the data obtained for 63-hydroxycortisol by the MSSMET program. Also shown are data for a peak which occurred at retention index 3290. This peak exhibited the same ion currents as the major peak at R.I = 3313, was found in both the standard and the urine samples, and most likely is 6a-hydroxycortisol. These data were obtained from the packed column runs and were normalized to the internal standard and urinary creatinine. 63-Hydroxycortisol was not present in high enough concentration to be observed in the capillary column GC/MS analyses. These data are included to demonstrate the sensitivity of the automated reverse library search. Although 63-hydroxycortisol is a minor component of the urinary steroid mixture, this compound was found in approximately half of the samples analyzed and the averages obtained from these analyses were consistent with data obtained from the capillary column 00 with FID analyses. Since this metabolite of cortisol is a minor component of the steroid mixture, the procedure utilizing capillary column G0 with FID detection proved to be a more quantitative method of analysis, since this method had the required sensitivity (see Section II). Figure 25. 145 Electron impact mass spectrum of the methoxime-trimethylsilyl derivative of 63-hydroxycortisol. The molecular ion is m/z=724. The m+-31 ion (m/z=693) and subsequent losses of 90, 2X90 and 3X90 are very characteristic of methoxime- trimethylsilyl derivatives of steroids. 6-beta -hyd roxycortisol - MO -TMS 513 393 363 499 586 603 2;. 3:1 1:1 ——-——-——-- 693 724 96 490 146 Table 9. Determination of 68-hydroxycortisol by agtomated reverse library search of selected mass chromatograms . Retention Index2 (1012 33.13 3.21 Controls (n=13) 12.4 1 14.2 1.2 1 3.3 PBBs (n=14) 23.7 1 24.0 1.8 1 3.4 Phenobarbital (n=6) 118 1 233 17.6 1 45.8 1 Data normalized to the internal standard (in ng) and urinary creatinine. Data were obtained from packed column runs since 63-hydroxycortisol was not in high enough concentration to be observed in the capillary column GC/MS analyses. 2 The peak at retention index 3313 was the major peak obtained for the 63-hydroxycortisol standard. The minor peak at 3290 had an almost identical mass spectrum and was probably 6o-hydroxycortisol. 147 The urinary concentrations of 63-hydroxycortisol in the control and phenobarbital groups are plotted in Figure 26. All subjects were non-smoking adult males. Data are expressed as ug of 63-hydroxycortisol/mg creatinine (a relative response factor for 63-hydroxycortisol, and cholesteryl butyrate had previously been determined allowing conversion to actual amounts of compound). The levels of urinary 63-hydroxycortisol were greatly elevated in some, but not all, of the phenobarbital exposed individuals. It should be noted that the standard deviation of the mean was nearly as great as, or greater than the mean in this group, which was the same pattern observed by other investigators (Saenger gt gl., 1981; Roots gt gl., 1979; Ohnhaus and Park, 1979). Figure 27 shows the urinary 63-hydroxycortisol concentrations in the P885 and control groups (again, non-smoking adult males). The concentrations of urinary 63-hydroxycortisol were significantly elevated in the PBBs group; t-test, p<0.05. This observation was not surprising in view of the literature described above. However, the effect is best described as "mild“ since no dramatically increased concentrations were noted. Effects of PBBs exgosure on the urinary steroid metabolicgprofile in humans. Urinary concentrations of the major steroid components were most easily and acurately determned by capillary GC with FID. Data for the P885 and control groups for the major urinary steroids have been Figure 26. 148 Dot plots of urinary concentrations of 63-hydroxycortisol in phenobarbital and control groups. Data are for non-smoking healthy adult male subjects and are expressed as 09 of 68-hydroxycortisol/mg creatinine. Data were obtained using a 60 m DB-l fused silica capillary (narrow bore, thin film). 2.70-* ' .6 .. _. . .5 - - .4 - .1 o .3 _ . - O .2 - .. ' O 0.. .1 ‘ ‘ O 3 CONTROLS PHENO BARB ITAL ug of 6-beta -hyd roxycortisol / mg creatinine non-smoking adult male subjects Figure 27. 150 Dot plots of urinary concentrations of 63-hydroxycortisol in PBBs exposed and control groups. Data are for non-smoking healthy adult male subjects and are expressed as 39 of 63-hydroxycortisol/mg creatinine. Data were obtained using a 60 m DB-l fused silica capillary column (narrow bore, thin film). CONTROLS PBBS . C . O . .0 . O. _ C C . . . . ... ... . C C. 4 . '. . ug of 6-beta -hyd roxycortisol I mg creatinine non-smoking adult male subjects 152 tabulated in Table 10. The data were generated using a 60 m DB-1 fused silica capillary column. Groups were compared for statistically significant differences by t-test with the level of significance at p<0.05. No differences were noted in the excretion rate of the major metabolites of testos- terone (androsterone, etiocholanolone, DHEA, androstene- dione). 113-Hydroxyandrosterone, 113-hydroxyetiocholano- lone, 11-keto-androsterone and 11-keto-etiocholanolone are the major urinary steroids formed from 17o-hydroxy C-21 steroids by oxidative cleavage of the C-20,21 side chain. Of these particular steroids, 11-keto-etiocholanolone was increased in the PBBs group. The major urinary metabolite of progesterone, pregnanediol, was increased almost four- fold in the PBBs group. Certain of the major urinary metabolites of cortisol and corticosterone were also increased in the PBBs group. These steroids were THE, THF and a-cortolone. In general, the minor C-21 urinary steroids showed no tendency to be different. However, THB and THDOC were elevated in the PBBs group. Three unknown steroids, characterized by methylene unit retention indices, were also increased in the PBBs group. These data are tabulated in Table 11. The peak at R1 = 3365 was most interesting in that the peak occurred in all but two of the PBBs subjects but was found in only two of the 13 control subjects. Although the urinary levels of the major steroids and well separated minor steroids were more accurately 153 Table 10. Human urinary steroid metabolic profiles of non-smoking male subjects: Average plus standard deviation, normalized to the internal standard and urinary creatinine. amount/mg creatinine2 Control PBBs STEROID1 "76:13) (3:14) Androsterone 1.40 1 0.70 1.60 1 0.83 Etiocholanolone 1.30 1 0.82 2.00 1 2.00 113-hydroxy-A 0.90 1 0.50 1.30 1 0.60 113-hydroxy-E 0.80 1 0.70 0.80 1 1.00 DHEA 0.50 1 0.80 0.70 1 1.00 11-keto-andro. 0.20 1 0.10 0.20 1 0.10 11-keto-etio. 0.30 1 0.30 0.50 1 0.40* Androstenedione 0.30 1 0.20 0.40 1 0.30 Pregnanediol 0.14 1 0.04 0.50 1 0.70* Pregnanetriol 0.50 1 0.20 0.60 1 0.30 THE 3.00 1 2.00 3.00 1 2.00* THF 1.90 1 0.80 3.00 1 1.00* allo-THF 1.60 1 0.90 2.00 1 1.00 a-cortolone 0.90 1 0.30 1.20 1 0.40* 3-cortolone + 3-cort0l 0.70 1 0.30 0.90 1 0.30 a-cortol 0.40 1 0.20 0.50 1 0.20 THB 0.21 1 0.07 0.40 1 0.40* allo-THB and allo-THA 0.50 1 0.30 0.60 1 0.30 allo-THE 0.30 1 0.10 0.40 1 0.10 THDOC 0.06 1 0.05 0.12 1 0.09* THS 0.20 1 0.10 0.30 1 0.20 allo-THS 0.06 1 0.09 0.10 1 0.09 1 Abbreviations are listed in Table 6. 2 Relative response factors for MO-TMS derivatives of these steroids was not calculated. However, most of these values will be close to 1.0. Thus, values listed are very close to the actual amounts in ug/mg creatinine. * Significantly different (p<.05) 154 Table 11. Unknowns elevated in the urine of PBBs exposed human subjects as determined by capillary BC with FID. Average 1 standard deviation1 amount/mg creatinine Control PBBs R.I.2 (n=13) (n=14) 2626 0.06 1 0.03 0.18 1 0.25* 3180 0.12 1 0.05 0.20 1 0.12* 3365 0.02 1 0.05 0.10 1 0.08* 1 Normalized to the internal standard and urinary creatinine. 2 Each compound was identified by these characteristic retention indices. * Significantly different (p<.05). 155 determined by capillary BC with FID, there were a number of other steroids that were detected by GC/MS/DS. These included compounds in the MSSMET library where retention and spectral information were obtained from standards, compounds where retention and spectral information were obtained from other investigators and compounds where retention and spectral information were obtained directly from GC/MS analysis of the urinary milieu (i.e., entered in the MSSMET library as unknowns). These data are displayed in Table 12. Values are the integrated area of the designate ion for each steroid divided by the integrated area of the designate ion of cholesteryl butyrate (m/z = 368) times the amount of cholesteryl butyrate added (in mg) divided by the mls of urine used and the concentration of creatinine (in mg/ml). Data shown are the average t 5.0. for controls (n=13) and P335 (n=14) subjects. Also shown are the averages t 5.0. for the phenobarbital treated subjects (same subjects for which Gs-hydroxycortisol concentrations are shown in Figure 26). Since these are for the most part minor urinary steroids, the data shown are for the packed column GC/MS/DS analyses. Also shown are values for THE and THF, which are included to demonstrate that the GC/MS/DS gave comparable results for the major urinary steroids as GC/FID (Table 10). Most of the steroids and unknowns listed in Table 12 are in very low concentration in urine. For this reason, these compounds were often not detected by automated reverse library search of selected mass chromatograms. As an 156 o.¢ H o.H om H om o.m A o.~ Amcouufl-mcmumogucmumuaxoeuxg_LuuwH .mH.amv H gown .xoccx;_vumfi.aoa ma cm 1 cm OH 1 01 cm 1 OH o:o-o~-a=1=macg-mm-»xoeu»;_u-am.am 11 ow « om OH H oH cm H oH economumcmcmmgqumm-xxogu»;_uuamfi.am NH cm 1 OH com 1 OOH om H on A_o.1u-1~H .amH.mmum=mumoeucnumv _e_gumcmumogu=< 0H cm H 0H m H N o.m « o.H Amco-~H-mcmumogucmum-»xoguxg_Lu -aoH.1mH.1mv wu=_ ummoqu _ouwagunocmga can vmmoaxm mama .mpocucou :_ mv_ogmum meagre: cmsaz .NH «_nmh 157 OO ON OO OO OO OO HOO1O-1ON.1NH.ON-OOOOO11O-O NO OH O.N OO OO OH OH OOO-ON-OOOOOOLO-1O-OXOLOOO_O-aOH.ON OO 0.0 0.0 Om ON OH OH HOOO-ON-OOOOOOLO-OO-OXO1OOO_O namH.umv acoHocmcmmNQAxocuxguamH me 0.0 0.0 0.0 O.N O.N O.H OOOOO11O-O-OXO1OOOO.H-aON.aOH.Om OO OOH OO OO OO OO OO OOOOOO1O-OO-OxoeoxgmeumO-HN.aON.OHH.aO NO ON ON OO Om OO ON OOOOOOLO-OO-OxO1OOOOLHOH-HN.aON.OHH.aO NO ON OH ON OH OH NH OOOOOOLO-OO-OXO1OOO_LH-aON.OHH.aO ON ON OH OH OH ON ON HOOO-HH-OOOO11O-OO-O1O1OOOO.N -aON.aNH.aOO HOH111111O11O-OOO¥-HH ON OOO OOO OO OO OO ON HOHep-aON.aOH.aO-OOOOO11O-OO ON OH 0.0 OO ON OH O 11°-HH-OOOOO111-OO-OXO1OOOOO-aON-aO ON OH O.O OH 0.0 O.N 0.0 OOO-HH-OOOOOOLO-aO-OXO1OOO_O-aON.aO ON OOO OON OOH OO OO OO OOO-ON-OOOOO111-1O-OxO1OOOHO-aOH.ON NN 0.0 O.¢ HH OH O.H O.H HOOOHLNOLOOO -HOHOO.O.H-H1O1OOOH1N-ONH.1OH.OO HOOLOON HN O.N O.N OO ON 0.0 O.N HOOO-NH-OOOOOO1OOO-O-O1O1OOOOLN-OH .OH.OOO N 1111 .OOOO-OxO1OOOHO-OH.aOH ON HOuOO HOHuOO HOHuON O z 1 z 1aOst Hmuwngmnocwca mama Opogucou ucaoaeou HOOOOONOOOO NH OHOOH 158 0.0 1 0.0 ON 1 OH OH 1 OH N1HHZO 1HN O.O 1 O.1 O.1 1 O.N OH 1 OH NNHHZO NHN OON 1 OON ON 1 ON ON 1 ON NNHHZO NHN OON 1 OOH ON 1 OO ON 1 ON NHHHZO HHN OH 1 O.1 NN 1 O.N O.OH 1 O.1 NHHHZO OON OON 1 OOH OO1 1 OOH OH 1 1H NOHHZO OON O.O 1 O.O O.N 1 O.N O.H 1 1.O NHNZO ONH O.1 1 O.N O.N 1 O.N O.H 1 O.O NONzO ONH OOO 1 OO1 OO1 1 OOO OO1 1 OO1 NONZO 1NH O1 1 ON ON 1 ON ON 1 ON NONZO NNH O.O 1 0.0 ON 1 O.1 0.0 1 O.O NNNZO NNH ON 1 OH OOON 1 OOO OOH 1 OO NONzO HNH OO 1 OOH OON 1 OON OON 1 OON NONZO ONH O.O 1 O.N OON 1 OO ON 1 OH N1N2O OHH OO 1 O1 OOH 1 O1 ON 1 OH 1:1-ON-1OOOOmaOOxO1Oas111-11H.N.H OO OO 1 ON OON 1 OOH O1 1 ON H11111-ONH.1OH.OOH.1N-1111111OO1-O OO 11111 H1H1=O HNHuOO O z < z 111112 NONNOLOnocmOO Oman mpocucou uczoaeou Auw:=_u=ouv NH mHnON 159 .OmNHNE HLOOHL: mg» 10 mmeHmcu mz\um Eogw aHuumLHO cm:_muno mew: OOHNOELomcw Huguumam vcm cowucmumg omen: Oczocxcs N .Ouownnsm umuumgu HNNNOLOnocmca mg» 101 can mazoca HcHucv mama gmN; new .HmHucv mama 30H LO1 .o.m 1 came mg» ago czogm ONO: .HHs\me cwv «OOONNNmLO 1o cowumgucmucoo mgu can now: mOOL: mo OHE on» On umu1>Nu .Hm: ONV canto mumgxuan ngmummHogu mo “cacao oz» Omawu .Hmmm u ~\EV muwcauzn ngmuOmHogo 1o :01 mpmcmNOmu msu mo mmgm umumcmmHON mg» On umu1>1u chocxca gov uwogmpm zoom go; OOO mpmcmNOmu mg» 1o ngm umumcmmucw as» mgm OmOHO> H H111111OOOV NH 1H111 160 example, estriol (3,16a,17B-trihydroxy-1,3,5(10)- estratriene one of the major urinary metabolites of 173-estradiol in humans) was found in 8 out of 14 of the high PBBs subjects and in only 4 out of 13 of the low PBBs groups. Estriol was observed to increase in the P385 (x = 0.8 t 1.4 in the low PBBs group and X = 5.9 t 11.0 in the high PBBs group). Also, all estriol values in the high PBB group were higher than the highest value recorded in the low PBBs group. The data for estriol is typical of most of the data in Table 12 because this compound was either below or near the detection limits of the analytical system. For these analyses, the values determining whether a compound will be printed in the found file were set purposely low. This, of course, increased the possibility of obtaining “false positives“. However, for this particular analysis of the GC/MS data, the object was to detect possible differences only, since most of these compounds were at or below the level of detection. Obviously, more sensitive methods of analysis would have to be employed to confirm or refute any differences noted when using an automated reverse library search for quantitative analysis of minor components of the urinary steroid milieu. Inspection of the data in Table 12 revealed that a number of compounds were apparently increased in the P885 exposed individuals. Most interestingly, many of these compounds were 16a-hydroxy steroids. For example, large increases in the excretion 161 of androstenetriol (5-androstene-3B,16o,173-triol) 16a,18- dihydroxy-DHEA, estriol, 33,16a-dihydroxy-Sa-pregnane-ZO-one and 5-androstene-33,155,16a,17B-tetrol were seen in the high PBBs group when compared to the control group. Other apparent differences were seen for UN24 and UN110. UN110 had the same retention index as the first compound listed in Table 11 (2626) and these may, in fact, be the same compounds. Mass spectral interpretation of UN110 indicated this compound might be a hydroxylated metabolite of progesterone. DISCUSSION The subjects. The data presented above indicate that PBBs exposed individuals may have altered excretion rates of certain urinary steroids compared to an appropriate non-exposed (or low level of exposure) group. These urinary samples were obtained through the Michigan Department of Public Health (MDPH), Division of Environmental Epidemology from individuals enrolled in a study examining possible long-term health effects of accidental PBBs exposure. Factors that were considered in the selection of subjects have been described above. What emerged from this selection process was two groups of apparently healthy male subjects who did not smoke and had no known, or reported, medical consideration that would obviously necessitate the removal of them from this study. Of course, one must trust the honesty of the respondents and this is not always a correct assumption. For example, the questionnaires were filled out by employees of the MDPH who interviewed each participant in the study. Under the interview conditions, certain subjects might have felt inhibited in providing a totally accurate medical history. For example, an individual who professed to be a non-smoker might in truth have an occasional cigarette but be reluctant to reveal this information. 162 163 However, the relatively large number of subjects selected for each group will tend to overcome the few instances of inaccuracy which most likely occurred in the interviewing process. For the most part, it is this author's belief that the high and low PBBs groups were closely matched with respect to those factors known to effect steroid hormone excretion. GB-Hydroxycortisol. Data comparing the urinary levels of 63-hydroxycortisol in the high and low PBBs groups were one of the more interesting aspects of this study. As pre- viously stated, a number of drugs known to be classical inducers of hepatic P-4SO microsomal mixed function oxidase system have been shown to increase the urinary levels of GB-hydroxycortisol in a dose-dependent fashion, with the highest doses of various drugs causing a many-fold increase in the average for the treated group. With increased exposure (or dose), there was also a concomitant increase in the standard deviation of the group mean. If hepatic micro- somal mixed function oxidase activity in individuals with "high" plasma levels of P835 was induced by P385 to the extent seen following administration of a large or maximum dose of one of the classical P-450 type inducers, then large increases in the excretion of Ga-hydroxycortisol, at least in some of the experimental subjects, should have occurred. Although the mean of the high PBBs group was significantly elevated over the low PBBs group, the increase was less than two-fold and no individual had a greatly elevated 164 concentration of 68-hydroxycortisol. Therefore, it follows that the PBBs-exposed individuals were certainly not experiencing the same degree of stimulation of hepatic P-450 microsomal MFO enzyme stimulation that was seen following large or maximal doses of the drugs previously mentioned. What can be said is there was an apparently "mild" stimulation of these enzyme systems in the high PBBs groups similar to what has been reported to occur following "moderate" doses of the above-mentioned drugs. This "mild" induction of hepatic microsomal mixed func- tion oxidase enzyme systems was not surprising in view of the literature. First, the inductive affect of P885 on hepatic P-450 enzyme systems is well established in experi- mental animals (Dent gt al., 1976a, 1976b; McCormack 35 al., 1978; Newton 33 _1., 1980, 1981) and would be expected to occur in man. Second, reports on workers exposed to DDT and endrin have also indicated a mild stimulation of 63-hydroxy- cortisol excretion. Poland £5 31. (1970) studied the effect of intensive occupational exposure to DDT on drug and steroid metabolism. DDT (1,1,1-trichloro-2,2 bis(p-chloro- phenyl)ethane) is similar to PBBs with respect to effects on hepatic microsomal MFO enzyme systems and storage in body fat tissues. In Poland's study, the concentration of DDT and DDT-related compounds was 20 to 30 times that of the control population. They reported that the serum half life of phenylbutazone (also a test for hepatic P-450 microsomal mixed function oxidase induction) was 19% lower in the 165 factory workers exposed to DDT and that urinary excretion of 63-hydroxycortisol was 57% higher in this group. Both of these differences were significant at the p<0.0l level. Increased excretion of GB-hydroxycortisol (2-3 fold; n=8) has also been reported to occur in workers exposed to endrin (Chamberlain, 1971). Both of these studies, involving exposure to fat soluble polyhalogenated organics similar to PBBs, report an effect on 6B-hydroxycortisol very much in line with that seen in the present investigation. Table 13 summarizes some of the more important metabolic consequences of induction of hepatic microsomal mixed function enzyme systems. Although a number of consequences are harmful (or toxic) under certain situations, there are also potential beneficial consequences. Thus, it could be misleading to describe an apparent stimulation of hepatic cytochrome P-450 mixed function oxidase systems by P385 in the exposed p0pulation as a toxic affect. Urinary Steroid Metabolic Profiles. The urinary steroid metabolic profile of the high PBBs group showed some potentially important differences. With the exception of pregnanediol (the major urinary metabolite of progesterone) and possibly estriol, all of the steroids elevated in the high PBBs group are of adrenal origin. To reiterate, these steroids are Il—keto-etiocholanolone (formed by oxidative cleavage of the sidechain of a 17a-hydroxy-20-one C-21 steroid), THE, THF, a-cortolone, THB and THDOC. If there are, in fact, elevated concentrations of urinary steroids of 166 Table 13. Possible consequences of induction and/or inhibition of liver microsomal enzyme systems. 1) 2) 3) 4) Increased metabolism of endogenous substrates Decreased metabolism of endogenous substrates Increased levels of minor metabolites of endogenous substrates or appearance of a new metabolite Shift in dose-reponse curve to drug therapy a) Attenuated therapeutic and/or toxic response to drug therapy b) Increased therapeutic and/or toxic reponse to drug therapy Shift in dose-reponse curve to toxin exposure a) Attenuated response to accidental exposure to a toxin b) Increased response to accidental exposure to a toxin 167 adrenal origin in the high PBBs group, this may indicate an effect of P885 that is unrelated to stimulation of hepatic cytochrome P-450 mixed function oxidase induction since total urinary 17-hydroxycorticosteroids in humans does not change following administration of phenobarbital or rifampicin (Saenger gt al., 1981; Ohnhaus and Park, 1979). Thus if PBBs do effect adrenal function in humans, an alternate explanation of the toxicodynamics of this effect must be found. Indeed, PCBs, compounds similar in chemical and toxicologic properties to PBBS, have been reported to elevate corticosterone and cortisol levels and increase adrenal size in rodents and rhesus monkeys (Sanders gt al., 1974; Wasserman _t._l., 1973; Barsotti and Allen, 1975). If PBBs do in fact stimulate adrenal function in exposed humans, the effect may be of toxicological significance. Statistical considerations. There are certain statis- tical problems associated with analysis of data of the type displayed in Tables 10-12. Although there were statistical- ly significant differences noted in seven out of the 22 steroids listed in Table 10, the chance of finding differ- ences between any two groups does increase with the number of comparisons made. For example, if 20 separate measure- ments are compared and the level of significance is arbitrarily set at p<0.05, one would expect to find at least one significantly different group of values by chance alone, assuming the measurements are stastically independent. Thus, a simple t-test, or a similar test, are not the most 168 Figure 28. Plots of Student's t-statistic when urinary steroid data are randomly reassigned. Compounds; are ordered by value of t-statistic (top plot). Six values were randomly picked from each group and reassigned to the other experimental group. The statistical comparisons were then repeated and the 10 largest values plotted. Data shown are for 18 replications of this process. 3 12 2 6 7 5 7 3 1 5 9 10 O a; c.” N N '1! O N O. u 0 U “W“ b 1 13 6 5 4 2 1O 13 5 1 6 3 O 1 3 5 11 9 7 4 12 13 2 3 no we on. use w . can» «a n15; HHN)“ H O 0 .fi .— b “_- t N P P p p P p b > D b p 9 14 1O 3 2 1 3 13 T 10 4 11 mun“ 1 16 O 3 2 13 13 3 2 12 O 10 HHHH Q S 7 11 3 2 6 9 2 1 3 5 NH!“ ‘ 2 8 5 1 3 — ’— b ( l 10 11 9 16 13 5 1O 1 2 5 9 6 ‘4..- N AAAAAAA ALA 1 h 1 b AMAA-AAAA A AAA 13 12 1 5 2 1O 8 O 7 12 1 3 mm ”a 0 O ... on w J 01.. 1 3 10 O “m E ( l 170 appropriate methods to use when comparing multiple variables between groups, as is the situation in metabolic profiling. However, there are tests to ascertain the level of signifi- cance that can be placed on these data as a whole. One test applied to the current data involved ranking each compound by its level of significance using the Student's t-statistic (LePage, 1983). This procedure involved first calculating the level of significance for each compound using the t-statistic, which is simply the difference between sample means divided by the standard error of difference of sample means and is calculated as follows: Xl’x2 t: 2 2 s-+s- X1 X2 where 71 and 72 are means for groups 1 and 2 and ga. and 5%? are standard errors of the means for groups 1 and 2. The absolute values of the ten most significant values (10 largest values) were tabulated in order of increasing significance. Bar graph plots of these data are shown at the top of Figure 28. Data are plotted with increasing t- statistic from top to bottom. Following this calculation, six subjects were randomly picked from the control group and reassigned to the high PBBs group and six subjects were randomly picked from the high PBBs group and reassigned to the control group. Following random reassignment of subjects, t-statistic values were again calculated for each compound. The absolute values of the ten most significant 171 values were tabulated in order of increasing significance. This process (randomization of the data and calculation of a new set of t-statistics) was then repeated a total of 24 times. Randomization of the data will have the tendency to attenuate differences occurring in the original data set although some greater differences will occur by chance alone with enough replications of the process. Thus, the relative number of times the sum of the t-statistics is greater than the original data set is a measure of the probability that the original distribution of means could have occurred by chance alone. From this type of analysis of the data, the level of significance for the group as a whole was placed at p