—-vv A. .. EC??- NS .11 0F Manx-mam out =32 ANDMAMMALIAN nss H¥¥£T LEO , . o . (($39 Pm.” ,.. 7 us. am. {Emma GAN>JKNEUNWER$TY T SAMUEL ALB " 1971 Thesis for the MBCH .npt Satin... n14. , .23.:- ... k i 5:4? mg... L: [\l' I x i . . v V . . ., , . 5.9.04 A 0'. a? .~ BRA l\\"l\l\\\'\lll\ll\‘illlfllfilll\ ll“ ”\/ \\\ W . . W RY ’ 3 1293 10539 4088 LIBRA ..MichiganStaw -- University This is to certify that the thesis entitled EFFECT OF MALATHION 0N SELECTED INSECT AND MMIALIAN TISSUE CUL'IURES presented by Albert Samuel has been accepted towards fulfillment of the requirements for _m.n.__ degree in 11119111911233. ,/Q ' \ C 5"W%}W7ZQ é/ai'x/ Major professor Date //"/'-7/ 0-7639 F“ ”1.- l‘ 1‘ Ike! *1 q " . v-\ i.\\ ABSTRACT EFFECT OF MALATHION ON SELECTED INSECT AND MAMMALIAN TISSUE CULTURES BY Albert Samuel The effect of malathion (S-[1,2-bis-(ethoxycarbonyl)- ethyl]0,0-dimethyl phosphorodithioate) on cell cultures of insect and mammalian origin was studied with regard to DNA, protein, and RNA syntheses and population increase. Insect cell groups in general showed twice as much resis- tance to malathion as the mammalian cell group. Cyta- pathological effects seemed to appear after the cells had undergone a certain degree of inhibition. Absence of malaoxon in the "malathion intoxicated" cultures and the inability of malaoxon (up to 20 ppm) to produce any in- hibitory effects indicated that ”malathion" effect was not caused by malaoxon. Contributions to malathion toxicity by impurities was deemed insignificant, though possible contribution to toxicity by some metabolite was not elim- inated. The first malathion induced biochemical lesion was suspected to have occurred in the protein synthetic Albert Samuel process which in turn led to inhibition of DNA synthesis. High sensitivity of the cells to malathion with regard to mitotic arrest was noticed. The mitotic arrest seemed to have occurred before the M phase. The net effect of the inhibitions led to premature "aging" of the cells and cell death. It was found that NAD enhanced the hydrolysis of malathion in cell cultures, probably by increasing carboxy- esterase activity. NAD also enhanced the synthesis of DNA, probably because of the increased hydrolysis of malathion. The value of insect fat body cell cultures and primary embryonic cell cultures were evaluated by using cells obtained from house fly larvae. The fat body cells found not very useful for direct studies on cell division related processes. Embryonic cell cultures, as indicated especially by DDT metabolism, was found to have significant value in pesticide resistance studies. EFFECT OF MALATHION ON SELECTED INSECT AND MAMMALIAN TISSUE CULTURES BY Albert Samuel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of .DOCTOR OF PHILOSOPHY Department of Entomology 1971 To my parents ii ACKNOWLEDGMENTS I am very grateful to Professor R. A. Hoopingarner and sincerely thank him for help in selecting this pro- ject, inspiring guidance, and encouragement at all times. I sincerely thank Dr. J. E. Trosko for providing me with the mammalian cell cultures and his advice. I thank Dr. R. E. Monroe for his constant help. My thanks also go to Dr. R. L. Fischer for his helpful suggestions, and Dr. Dean Branson of Dow Chemical Co., Midland, Michigan for providing me with the Antheria eucalypti cell culture and his helpful suggestions. I wish to express my thanks to Mirium Isoun for her technical assistance in culturing the tissues and to Larry Besaw for his technical assistance in DDT metabolism studies. To my wife, Jean, for her understanding, encourage- ment, willing assistance at all times, and for typing the manuscript goes my special thanks. My final thanks to the Chairman of the Department of Entomology, Dr. G. E. Guyer, for providing me the oppor— tunity to obtain education in this department. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . INTRODUCTION 0 O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . I. II. III. IV. V. Past Attempts at Long Term In Vitro Tissue Culture . . . . . . . . . Serum in Tissue Culture Media . . . Morphology and Behavior of Cells in Tissue Culture . . . . . . . . . The Application of Tissue Culture Methods in Pharmacological Investigations . . . . . . . . . Malathion . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . I. II. III. IV. Cell Cultures . . . . . . . . . . . Studies on the Interaction of Malathion and Malaoxon with Cell Cultures . DDT Metabolism in Musca domestica Embryonic Cell Cultures . . . . . General . . . . . . . . . . . . . . iv Page vii viii 13 25 30 30 36 46 47 Page “SULTS O O O O O O O O O O O O 0 0 O O O O O O O 4 8 I. Antheria eucalypti Cell Cultures . . . . . 48 II. Studies on Embryonic Cells of Musca domeStica 0 o o o o o o o o o o o o o o 56 III. Studies on the Fat Body Cells of Musca domestica Larvae . . . . . . . . . . . . 59 IV. Studies on Human Amnion Cells . . . . . . 61 V. Studies on Chinese Hamster Cells . . . . . 63 VI. Comparative Study on the Effect of Malathion, Malaoxon, and "Impurities" Found in Malathion and Malaoxon Compounds . . . . . . . . . . . . . . . 74 VII. The Inhibition, by Malathion, of Thymidine-3H Incorporation by Cells as Affected by NAD and NADH . . . . . . . . . . . . . . . . . . 79 VIII. Studies to Determine Whether Malathion was Converted to Malaoxon in the Tissue Cultures . . . . . . . . . . . . 79 IX. DDT Metabolism in DDT-R and N—R Embryonic Cell Cultures . . . . . . . . 82 DISCUSSION . . . . . . . . . . . . . . . . . . . 85 I. "Malathion" Toxicity . . . . . . . . . . . 85 II. Interrelationship Between DNA, RNA, Protein and Cell Division Inhibitors O O I O O I O O O O O O O O O 92 III. Mitotic Arrest and Cell Death . . . . . . 95 IV. The Effect of NAD and NADH on "Malathion Effect" . . . . . . . . . . . . . . . . 97 Page V. Musca domestica Fat Body Cell cuItures O O O I O I I O O O O O O O O O 99 VI. The Metabolism of DDT in DDT-R and N-R Embryonic Cell Cultures . . . . . . . . lOl SUMMARY . . . . . . . . . . . . . . . . . . . . . 106 LITERATURE CITED . . . . . . . . . . . . . . . . 110 APPENDIX . . . . . . . . . . . . . . . . . . . . 125 vi LIST OF TABLES Table Page 1. Effect of Malathion on the Incorporation of Thymidine-3H Uptake by Antheria eucalypti Cells . . . . . . . . . . . . . 51 2. Effect of Malathion (75 ppm) on the Protein Synthesis of Antheria eucalypti Cells . . . . . . . . . . . . . 52 3. Malathion Effect on the Viability of Antheria eucalypti Cells Based on MorphologiCal Aspects . . . . . . . . . . 53 4. Histochemical Studies on 4-day-old Fat Body Cultures of Musca domestica Larvae O O O O O O O O C O O O O I O O O 6 l 5. Malaoxon "Impurities" Separation . . . . . 76 6. Radiometric Analysis of the Effect of NAD and NADH on the Hydrolysis of Malathion in Insect Tissue Cultures . . . . . . . . . . . . . . . . 81 7. Percent Areas of the Unknown Peaks, as Compared to Malathion Peak Areas, for the Different Cultures . . . . . . . 83 8. Conversion of DDT-#DDE in DDT-resistant and DDT-susceptible Flies . . . . . . . . 84 vii LIST OF FIGURES Figure Page 1. Growth Curve of Grace's Antheria eucalypti Established Cell Line . . . . . 49 2. Incorporation of Thymidine-3H into DNA by Antheria eucalypti Cells . . . . . 50 3. Antheria eucalypti Cells. "Normal" Cu1ture O O I O O O O O O O O O O I 0 O 0 S4 4. Antheria eucalypti Cells. Telophase Cells Shown . . . . . . . . . . . . . . . 54 5. Antheria eucalypti Cells. Anaphase and Prophase Cells Shown . . . . . . . . 55 6. Antheria eucalypti Cells. Malathion Treated (75 ppm) . . . . . . . . . . . . 55 7. Musca domestica Embryonic Cells. 1 Hour Old Culture . . . . . . . . . . . 57 8. Musca domestica Embryonic Cells. 8-10 Hours Old Culture . . . . . . . . . 57 9. Effect of Malathion on Thymidine-3H Incorporation in Embryonic Cell Cultures of Musca domestica . . . . . . . 60 10. The Effect of Malathion (25 ppm) on the Incorporation of Thymidine, Uracil, and Phenylalanine in Human Amnion Cells . . . . . . . . . . . . . . 62 11. Effects of Different Dosages of Malathion on the Thymidine-3H Incorporation in Chinese Hamster Cells . . . . . . . . . . . . . . . . . . 64 viii Figure Page 12. Effect of Malathion on t e Incor- poration of Thymidine- H by Chinese Hamster Cells During the First 6 Hours of Incubation . . . . . . . 65 13. Inhibition of Mitosis by Malathion in Chinese Hamster Cell Cultures . . . . 66 14. Chinese Hamster Cells. "Normal" Culture . . . . . . . . . . . . . . . . . 67 15. Chinese Hamster Cells. "Normal" Culture. 10 Hours Old Cultures . . . . . 69 16. Chinese Hamster Cells. Malathion Treated (30 ppm). 24 Hours After Treatment . . . . . . . . . . . . . 7O 17. Chinese Hamster Cells. Malathion Treated (50 ppm). 10-12 Hours After Treatment . . . . . . . . . . . . . 71 18. Chinese Hamster Cells. Malathion Treated (50 ppm). 24 Hours After Treatment . . . . . . . . . . . . . 71 19. Effect of Malathion and Malaoxon on the Multiplication of Chinese Hamster cells 0 o o o o o o o o ‘ o o o o o 73 20. Effect of Malaoxon (0-20 ppm; 10 % Pure Sample) on the Thymidine- H Incorporation by Chinese Hamster Cells . . . . . . . . . . . . . . . . . . 75 21. Effect of Malathion (Purified 100%), Malaoxon (Purified 100%), and "Impurities" on % Thymidine- H Incorporation by Chinese Hamster Cells . . . . . . . . . . . . . . . . . . 78 22. Effect of Cofactors NAD and NADH in Malathion Treated Cultures of Antheria eucalypti C3113 with regard to Thymidine- H Incorporation . . 80 ix Figure Page 23. Radio autography of Musca domestica Fat Body Cells . . . . . . . . . . . . . . 126 24. Musca domestica Fat Body Cells. Glutamic dehydrogenase . . . . . . . . . 127 25. Musca domestica Fat Body Cells. a-glycerophosphate dehydrogenase . . . . 128 26. Musca domestica Fat Body Cells. Isocitric dehydrogenase . . . . . . . . 129 INTRODUCTION During the past decade significant progress has been made in the development of tissue culture techniques and their application to a wide variety of biological problems. Work has been especially prolific in areas such as vir- ology, cancer research, and radiation biology. Increasing employment of in yit£9_tissue studies are noted in areas such as animal cell genetics, cell physiology, and bio- chemistry. Pharmacology is one of the areas in which such techniques are being increasingly initiated. Pharmacology is the study of action of chemicals on living things (Pomerat gt a1., 1954) and as such includes a wide spectrum of approaches. Our general area of inter- est in pharmacology is the interaction of pesticides and animal cells under in yitgg conditions. In 1945 Lewis and Richards studied toxicity of DDT to a variety of chick embryo tissues which is regarded as the first attempt in using tissue culture as a tool in pesticide toxicity studies (Litterst gt 31., 1969). In 1965 Gabliks and Friedman, determined LD '3 for a variety 50 of insecticides, including organophosphorus compounds, on several mammalian cell cultures (Gabliks, l965a,b; Gabliks and Friedman, 1965). Organophosphorus insecticides are in general cholin- esterase inhibitors (Heath, 1961; O'Brien, 1960, 1967). Malathion (S-[l,2-bis-(ethoxycarbonyl)-ethyl]0,0-dimethy1 phosphorodithioate) is one such organophosphate insecti- cide whose main mode of action is cholinesterase inhibi- tion, though not as potent as some other members of the group (Spiller, 1961). While the action of malathion and other organophosphorus insecticides on the nervous system have attracted much attention, the effects of such in- secticides on other biological systems have not been studied as much (Wilson and Walker, 1966; O'Brien, 1961). Using tissue culture as a tool, we investigated the following: 1. The interaction of malathion and cells of insect and mammalian origin with regard to biological systems other than the nervous system. 2. Metabolism of DDT by house fly embryonic cells of two different strains. Tissue culture studies in general fall into three categories: 1. DevelOpment of techniques to keep tissues in culture conditions for a desirable length of time. 2. Making direct observations on the cells or tis- sues as to their various morphological and behavioral aspects. 3. Studying the various aspects of interaction be- tween the cells and specific organic or in- organic materials individually or in combina- tion, introduced into the tissue culture system. As indicated earlier, our general area of investi- gation, tissue-pesticide interaction, for the most part falls into the third category. However, considerable work in the first two categories was necessary in order .for a better evaluation of the problem under investigation. This report includes all such work accomplished. LITERATURE REVIEW I. Past Attempts at Long Term In Vitro Tissue Culture. A. Vertebrate Tissue Culture. The first recorded instance of successful in yitgg tissue culture dates back to 1885 when Wilhelm Roux, the embryologist, successfully maintained the medullary plate of a chick embryo in warm saline for a few days (Paul, 1965). Attempts at long term culturing were later made by workers such as Jolly in 1903 and Beebe and Ewing in 1906. However, it is Ross Harrison's work in 1907 that is generally acclaimed as the first successful tissue cul- ture work (Paul, 1965; Harrison, 1969). The tissue ob- tained from the medullary tube region of the frog by Har- rison in aseptic conditions survived for some weeks and differentiated into nerve cells giving rise to nerve fibers. Frog lymph clot was used as the growth media. Later further advances in this field were made by Alexis Carrel and his school. They demonstrated the possibility of continuous cultivation of rapidly growing and dividing cells over long periods of time through sub-culturing them (Carrel, 1912; Carrel and Ebeling, 1922). Invaluable contributions were made in this field by Dr. Wilton Earle and his co-workers toward improving the techniques and methods (Earle, 1943a,b,c; Earle and Crisp, 1943; Earle and Nettleship, 1943; Nettleship and Earle, 1943; Likely 35 31., 1952). During the succeeding years many verte— brate cell lines were established and maintained by many workers. Some such lines are cartilage cells (Fischer, 1922), human epidermoid cervical carcinoma and normal cells (Gey gt 31., 1952; Gey gt 31., 1954a; Moore gt_al., 1955), epithelioid and fibroblast-like cells from different human organs (Puck gt_al., 1957, 1958), chick embryo cells (Morgan £2 31., 1950), and Chinese hamster cells (Yerganian, 1958, 1961; Chu, 1965). B. Invertebrate Tissue Culture: With Specific Reference to Insect Tissue Culture. In comparison to vertebrate tissue culture, in- sect tissue culture took to the stage much later and pro- gressed much slower. The first attempt at insect tissue culture was made by Goldschmidt (1915). During the years that followed attempts were made by many workers toward es- tablishing long term insect tissue culture. A list of such attempts are given by Day and Grace (1959). However, it was in 1958 that a successful attempt was made by Grace in propagating cells in_yi£§2_for a considerable length of time (Grace, 1958). He successfully maintained the cells of promethea moth (Callosamia promethea Drury) pupae in Vitro for over twelve months. In 1960 he established a strain of cells from ovarian tissue of Antheria eucalypti Scott (Saturniidae) (Grace, 1962) which is still being carried by sub-culturing. The cells were grown in a tis- sue culture medium to which antibiotics and plasma of Antheria eucalypti were added (Grace, 1962). The culture medium was later modified by Yunker 33 31. (1967) to con- tain whole egg ultrafiltrate (10%), fetal calf serum (7%), and bovine albumine fraction V (1%) in lieu of the insect hemolymph. The doubling time of the adapted cells in this modified medium was approximately 2.5 days. Initial con- centration used was approximately 40,000 - 60,000 cells per milliliter (Yunker gt 31., 1967). Presently there are twelve insect cell lines de- rived from Lepidoptera, Diptera, Hemiptera, and Blattaria in existence. Details of the cell lines are given by Brooks and Kurtti (1971). The delayed beginning of insect cell culturing is attributed to the difficulties confronted in formulating suitable synthetic media for supporting long term insect cell cultures (Brooks and Kurtti, 1971). Although the method of approaching the formulation of an insect culture media was the same as for the vertebrates, appropriate modifications based on the hemolymph composition of the specific insect group were needed (Wyatt, 1956, 1961; Grace, 1962; Plantevin, 1967; Jones, 1966). The most re- markable feature of insect blood is the high concentration of amino acids and this is reflected in the formulation of the insect tissue culture media (Wyatt, 1956, 1961; Jones, 1966). Osmotic pressure and related K/Na ratio were some of the critical factors which had to be taken into con- sideration in formulations (Echalier gt gt., 1965; Jones, 1966; Moscona gt gt., 1965). II. Serum in Tissue Culture Media. Serum has been known to aid the growth of cells in tissue culture (Waymouth, 1965; Wallis gt gt., 1969; Brooks and Kurtti, 1971). In insect cell cultures insect hemolymph was generally used, but recently fetal calf serum has been successfully used in the place of hemo- lymph (Yunker gt_gt., 1967). However, the mode of action of serum in its cell growth-promoting capacity is not yet clearly known (Wallis gt_gt., 1969). According to Waymouth (1965) serum acts as a buffer and compensates for the inadequacies in the medium. Wallis gt gt, (1969) showed, with regard to their primary monkey kidney cell cultures, that the serum probably acted as an inhibitor of proteolytic enzymes. It inactivates the residual trypsin, used for dissociating the cells in the culture medium, and also the proteolytic enzymes synthesized by the cells. The growth-promoting nature of serum was shown to be related to the titer of the above enzymes. According to Michl and Svobodova (1969) the a-globulin fraction of the serum has growth-promoting capacity and it could serve as a source of high-energy phosphate. Their results were ob- tained on human diploid cell cultures. Jainchill and Todaro (1970) have recently shown that Y-globulin of calf serum promote cell growth in some mouse cell lines. How- ever, not all of their mouse cell strains responded. I Based on their experiment they suspected that the response of cells to the serum growth-promoting factor rested in the genetic make-up of the strains. It has also been shown by other workers in certain organ cultures that Bacto-Peptone or Proteose Peptone can be substituted for serum (Amborski gt_gt., 1970). III. Morphology and Behavior of Cells in Tissue Culture. A. Morphological Aspects. The two types of cells that usually occur in tissue culture studies are epithelial cells or epithelio- cytes, which are polygonal or somewhat rounded, and fibro- blast cells or mechanocytes, which are elongated and nar- row (e.g. viceral cells). These and other principle cell types, with regard to their morphology and origin, are discussed by Willmer (1965) and Paul (1965). Grace (1962) identified three distinct cell types in his insect cell cultures: 1. flat polygonal type with finely granulated cyto- plasm, 2. types which were round to fibroblastic in pro— file with distinct outline, and 3. types which had finely granulated cytoplasm and distinct outlines. Cells of fibroblastic and epithelioidal nature have also been reported by various other insect tissue culture workers (Echalier gt gt., 1965; Hirumi and Maramorosch, 1964a,b; Greenberg and Archetti, 1969; and Eide and Chang, 1969). In Hirumi and Maramorosch's (1964a) cultures of leaf- hopper embryonic cells, many of the fibroblastic-type cells became attached to the surface of the glass and had pseudo- podia with irregular outlines. In addition to such cells noticed by Eide and Chang (1969) in their house fly embry- onic tissue cultures, they also observed various other types of cells and significant size differences even in one type of cells. The difference in size is attributed to the age of the embryos (4-6 hours old). Though the cells seemed to aggregate secondarily into histologically identifiable groups, it was not possible to morphologically identify the tissues of origin. 10 B. Cell Contractions. Most of the above mentioned tissue culture work— ers observed rhythmically contracting cells. Hirumi and Maramorosch (l964a,b) attribute the rhythmic movements to the out-growths from large fragments of epithelial tissue, while Eide and Chang (1969) attribute it mainly to inner- vated muscles and to a lesser extent to non-innervated muscle cells. However, according to Paul (1965), the oligodendroglia cells pulsate rhythmically in £2.X£E£2 cultures while the muscle cells contract irregularly. C. Cell Reaggregation. Reaggregation of cells dissociated from embryonic tissues or organ rudiments in cell cultures is a commonly observed behavioral phenomenon (Moscona, 1965). The dis— persed cells are said to go through three general phases: (Moscona, 1965). 1. random aggregation which is called the primary aggregation, 2. secondary aggregation in which the cell groups are histologically identifiable, and 3. tertiary aggregates in which histo-differentiation and growth takes place. Eide and Chang (1969) reported that the behavior of their dispersed house fly embryonic cells followed the general pattern mentioned above. However, the course of cell ag- gregation can also be affected by numerous other factors 11 such as type of medium, developmental stage of cells at the time of dispersion, cell density, cell types, culture vessel surface, temperature, stability of the cultures, and factors such as serum protein (Moscona, 1965; Sato and Yasumura, 1966; Eide and Chang, 1969). Further de- velopment in such aggregates is said to depend on the ex- tent of differentiation they have undergone before dis- persal (Waddington, 1940; Moscona, 1965). D. Aggregation Counteracting Agents. The aggregation properties of the cells are counteracted by enzymatic and chelating agents such as trypsin and ethylene-diamine-tetra-acetic acid (EDTA) respectively (Morgan gt_gt., 1950; Lesseps, 1965; Paul, 1965; Moscona gt_gt., 1965). While trypsin acts by breaking the peptide bond between arginine and lysine and also by effecting the removal of mucoidal material around the cells (Jones, 1966), EDTA acts by removing the diva- lent cations that bind the tissues (Paul, 1965). Another agent that interferes with cell aggregation is puromycin which is an inhibitor of RNA-dependent protein synthesis (Moscona, 1965; Moscona and Moscona, 1966). The two first mentioned agents are widely used in dissociating cells from tissues for cell culture pur— poses (Moscona, 1965; Paul, 1965; Grace, 1962; Masters, 1965). It should be noted that tissue dissociation through these agents do cause damage to the cells. EDTA 12 treated tissues need to be strongly agitated in order to separate the cells and, as a consequence, cells are ex- tensively damaged. Since the damage caused by trypsin treatment seems to be comparatively much less serious than EDTA treatment, trypsin is more widely used (Moscona gt gt., 1965). However, according to Jones (1966) the extent of damage to the cells caused by trypsin is not yet clearly known. According to Bang (1966) under certain experimental conditions, trypsin could affect the viability of the cells. It should be further noted that trypsin does not work on all tissues; while embryonic organs are easily dis- sociated, adult organs with fibrous tissues do not easily lend themselves to trypsinization (Paul, 1965). E. Growth and Multiplication. Growth and multiplication which includes a com- plex of biological processes (Lehninger, 1970) are two of the fundamental behaviors of a living cell (Paul, 1965). Most tissue culture studies are centered around the abbve mentioned behaviors or properties of the cells either in general, or with specific reference to one or a few under- lying metabolic aspects such as protein, carbohydrate, lipid or nucleic acid metabolism. Valuable information regarding such works accomplished up to 1965 has been com- piled by Willmer (1965). Brooks and Kurtti (1971) have included a number of such studies in their review of in- sect tissue culture. 13 IV. The Application of Tissue Culture Methods in Pharmacological Investigations. One of the areas of biological research that employs tissue culture methods is the area of pharmacology. Ac- cording to Pomerat and Leake (1954), the scope of pharma- cological studies includes such aspects as: l. 2. 3. the dose-effect relations, time-concentration relationships, the absorption, distribution, metabolism and fate of chemicals introduced into the living tissue, the relationship between chemical constitution and biological action, and the mechanism by which chemical molecules intro- duced into living material react with the mole- cules comprising the cells of the living things. In general, it involves the various aspects of inter- actions between the cells and the various organic or in- organic chemicals introduced into the ecosystem of the tissue culture. The interactions are evaluated in terms of cytological effects, the effects produced on the cells which can be directly observed under the microscope, and biochemical effects. The biochemical effects generally fall into two categories: 14 l. The influence exerted by the chemicals on the various metabolic aspects of growth and cell division such as protein, lipid, carbohydrate, and nucleic acid metabolism which are studied through biochemical and physiological means. 2. The action of cells on the chemical(s) intro- duced in the system which is studied by analyzing the cell constitution or the medium for the chemical and its metabolites or the action of potential analogs on the cells through various biochemical analytical methods. A. Cytological Effects. Pomerat and Leake (1954) determined the relative toxicity of various therapeutic chemicals on certain tis- sues in culture. The rating scheme was based on the microscope observations of the distance traversed by emi— grating cells or nerve fibers, as the case may be. This criterion and several others, such as rounding up, granu- larity, disintegration, nuclear pycnosis, mitotic inhibi- tion, inhibition of outgrowth, vacuolation, and a few other cell-damaging properties, were used by many workers in the routine screening and forecasting of the therapeutic ac— tivity of desired chemical compounds (Loveless and Revell, 1949; Earle, l943a,b; Biesele, 1954; Duryee and Doherty, 1954; Fell, 1954; Gey gt gt., l954a,b; Lettré, H., 1954; 15 Lettré, R., 1954; Livingood and Hu, 1954; Murray gt gt., 1954; Pomerat and Leake, 1954; Pomerat gt gt,, 1954; Verne, 1954; Schneider, 1964; Echalier gt gt., 1965; Gabliks and Friedman, 1965; Kao and Puck, 1969; Masters, 1970; Mitsuhashi gt_gl., 1970a; Litterst and Lichtenstein, 1971). The action of vitamin A and hormones such as insulin and thyroxin on bone and cartilage tissue 12 gtttg_was micro- scopically evaluated (Fell, 1954). Criteria used were hypertrophy of the cells and differentiation of cartilage cells to bone cells. 1. Vacuolation. Formation of aqueous vacuoles in the cytoplasm of the cells was one criterion used in assessing the action of chemicals on cells tg_ztttg (Lettré, R., 1954). Such vacuolation occurred in his fibroblastic cells when organic amines or any substance splitting off ammonia, ammonium carbonate, adenosine or adenosine triphosphate (ATP) was added to the medium. Murray gt gt. (1954), in rating the toxicity of different azo compounds and normal purines to human tumor cells (glioblastoma multiforme), also used cytoplasmic vacuolation as one of the criteria. Rosenoer and Jacobson (1966) have reported a variety of drug induced vacuolation cases. They observed that the size of the vacuoles varied with the concentration of the drug tested. Different cell lines responded differential- ly to the same drug in terms of the general occurrence and 16 size of the vacuoles. For instance, pilocarpine produced vacuoles in HeLa cells but not in normal mouse cells. They suggest that vacuolation might be a mechanism by which cells try to cope with deleterious conditions, or might have some relation to the effect of drugs used on lysosomal membranes and related enzyme activity. Frost (1969) distinguishes between three different kinds of vacuoles: l. vacuoles accompanying degeneration, 2. phagocytic vacuoles, and 3. secretory vacuoles. Appearance of vacuoles in degenerating cells is due to the loss of control of cells over fluid balance, thus letting the water enter. Though vacuoles which are usually single and hyperdistended are said to be secretory in function, degenerative vacuoles also can be single and hyperdis- tended. However, vacuoles of the degenerating cells are many, evenly distributed, and do not displace the nucleus. Vacuoles of cell degeneration are observed in de— generating cells by many workers in their normal cell cul- tures not treated with any specific drug (Grace, 1962; Hirumi and Maramorosch, 1964a; Schneider, 1964). These observations indicate that vacuolations need not necessar- ily be specific to the action of any particular chemical, but a general indication of the onset of degeneration. 17 2. Granulation. Verne (1954) observed granulation in cytoplasm through infiltration of lipid granuoles. The origin of such lipid granuoles is not clear. According to the above author lipophanerosis could be the cause. However, cells that show such lipid degeneration did not necessarily die; they became normal when the medium was changed. Such lipid droplets along with vacuoles were also observed by Grace (1962) and Wyatt (1956) in their degenerating in- sect cells in culture. However, it seems that the granu- lation observed by Wyatt (1956) was not the same as those observed by Verne (1954) and Grace (1962) since Wyatt men- tions "granularity" and "fat droplets" as two distinct features that showed up in his degenerating cultured cells. 3. Nuclear Aberrations. Nuclear aberrations include abnormal nucleus, chromosomal aberrations, and gene mutations. A variety of chemicals are found to affect the mi— totic processes of the cells. These chemicals, which exert "toxic" effects on the nuclei without apparently injuring the cytoplasm, are called "mitotic poisons" (Loveless and Revell, 1949). A variety of related cri- teria are used in the evaluation of these chemicals. In the 1950's in the field of cancer therapeutics, the gener- al mitotic inhibition, nuclear pycnosis, and nuclear frag- mentation were used as criteria for evaluating desired 18 chemicals as to their effect on division processes of the cells (Murray EE.El-r 1954; Verne, 1954; Lettré, R., 1954; Biesele, 1954). Different azo compounds (Murray EE.El°r 1954), purine compounds (Biesele, 1954), mineral drugs such as mercuric chloride and organic toxic drugs such as potassium cyanide (Verne, 1954) were tested on tissue cul- tures derived from different mammalian organs. The effects varied quantitatively and with regard to the origin of the tissues. Geneticists and cytologists use more specific cri- teria, such as chromosome aberrations, inhibition of the formation of mitotic spindle, and gene mutations (Loveless and Revell, 1949; Kao and Puck, 1969). Legator and Jacobson (1970) grouped the different kinds of chromo- somal aberrations into that of numerical nature and struc- tural nature. The numerical aberrations include poly- ploidy and aneuploidy. The structural abnormalities in- clude gaps, breaks, deletions, fragmentations, and various forms of translocations, centromere rearrangements, and terminal blebs. There were also other forms of structural abnormalities such as chromosomal clumping and anaphase bridges noticed by other workers (Loveless and Revell, 1949). A wide variety of chemicals, including therapeutic drugs, nutritional additives, pesticides, other chemicals that are in common usage, and various industrial chemicals, are regularly screened by the above criteria (Wuu and 19 Grant, 1966; Chang and Klassen, 1968; Kao and Puck, 1969; Mauer gt gt., 1970; Krause, l970-personal communication). Inhibition of spindle formation is another form of "mitotic poisoning" (Verne, 1954) which has been exhibited by compounds such as colchicine (Loveless and Revell, 1949). Ostergren and Levan (1943) also have claimed such activity for various other chemicals. Short term tissue cultures have been used in detecting such chemicals by various workers (Verne, 1954; Lettré, R., 1954). Their observations, with regard to colchicine, indicated arrest of cell division at metaphase. The chromosomes were ob- served to clump or settle over the cell. Several chemicals have been found to produce single gene mutations in cells tg_ttttg (Kao and Puck, 1969). Their method took into consideration the induced nutrition requirements for the cell population and related popula- tion growth curve. It seems possible that organ cultures such as larval Drosophila eye-antennal disk cultures and the formation of pigments in this organ (Schneider, 1964) could also be used in detecting mutagenic properties of chemicals which will not visibly damage the chromosomes. B. Biochemical Effects. Tissue cultures, besides providing cytological parameters for screening desired chemicals, have come to be used as a useful biochemical tool. The advantages of using tissue cultures in biochemical areas have been 20 pointed out by Perlman (1968). Some of the biochemical aspects used as parameters in evaluating the activities of desired chemicals are protein metabolism (Wang gt_gt., 1970), carbohydrate metabolism, and nucleic acid metab- olism (Paul, 1959). Leslie and Paul (1954) studied the effects of insulin on glucose metabolism, DNA synthesis, and RNA synthesis. Further work along this line by Paul (1959) with new cell culturing methods yielded valuable information. Wagner and Roizman (1968) studied the effect of Vtggg alkaloids ("mitotic poisons") on rRNA and tRNA of human epidermoid cells. In pesticide research Lewis and Richards introduced the use of tissue cultures in 1945. They studied the ef- fects of DDT on chick embryo tissue cultures. With the increasing use of various chemicals, including agricul- tural pesticides and other agricultural chemicals, it has become necessary to have effective monitoring systems for detecting the environmental chemicals that might be a potential health hazard (Gabliks and Friedman, 1965; Litterst gt_gt., 1969; Hoopingarner and Bloomer, 1970). During the last ten years tissue culture has come to be used more and more in this field for the screening of such chemicals. In 1965 Gabliks (Gabliks, l965a,b; Gabliks and Friedman, 1965) in a series of experiments tested various 21 pesticides including DDT and malathion on HeLa cells. On the basis of observed cytotoxic effects (vide page 12) and the effect on gross protein synthesis, he established ID10 (inhibitory dose 10%), IDSO (inhibitory dose 50%), and TDSO (toxic dose 50%) for these various pesticides. Wilson and Walker (1966) investigated the toxicity of malathion and its various analogs with regard to cell population density. These workers, however, primarily used cytological parameters. Parameters of more funda- mental biochemical significance, with respect to growth and division of cells such as DNA, RNA, and protein metabolism, came to be used as appropriate analytical methods were introduced and improved (Hansen and Vande- voorde, 1966; Chung gt gt., 1967; Litterst gt_gt., 1969; Litterst and Lichtenstein, 1971). With the establishment of insect cell lines (Grace, 1962) and further advances made in the field of insect tissue culture in general, in- sect cell cultures also came to be used in such investi- gations (Mitsuhashi gt_gt., 1970a,b; Wang EE.Ei-' 1970). Effect on protein synthesis was determined by meas- uring the gross change in the general protein content of the cells using Folin-Ciocalteau method (Lowry gt gt., 1951; Oyama and Eagle, 1956; Wang gt_gt., 1970) and/or by determining the degree of radioactive precursor (amino acid) incorporation (Chung gt gt., 1967; Litterst gt 21°: 1969; Wang g 9.1-: 1970). 22 Incorporation of uridine-14C or uridine-3H into RNA was used in determining the effect on the synthesis of RNA (Chung gt_gt., 1967; Litterst gt gt., 1969; Wang gt gt., 1970). Chung gt_gt. (1967) and Litterst gt_gt. (1969) separated the RNA from other cell components of the cell before determining the extent of precursor incor— poration, Wang gt_gl. (1970), however, directly counted the radioactivity in the cells after only washing them. Effect of chemicals on DNA synthesis was determined by the amount of incorporation of thyamine-14C (Chung gt gt., 1967) or thymidine-14C or -3H (Painter, 1967; Litterst gt gl., 1969; Litterst and Lichtenstein, 1971). However, in Aedes aegypti cell cultures, Wang gt gt. (1970) could not use thyamine-14C since the mosquito cells in culture could not utilize this compound for DNA synthesis. For this reason thymidine-14C was used and found to be actively incorporated into DNA. With regard to using thymidine, Smets (1969) cautions against assuming the uptake of DNA precursors to quantitatively represent the incorporation of the precursor into DNA. According to the above author, precursors enter into an intracellular pool before it is incorporated into DNA. The amount of precursor in the pool and the amount incorporated were found to be different. However, Painter (1967) showed the uptake of thymidine to accurately measure the extent of DNA-synthesis. It should 23 be noted that while Smets used human kidney cells (T cells) in his experiments, Painter used HeLa cells. As indicated by the "central dogma" of molecular genetics (Lehninger, 1970), DNA, RNA, and protein syntheses are interrelated phenomenon. Besides this relationship based on functional contiguity, a relationship in terms of various phases of the life cycle of a cell in which components are synthesized has also been noticed. The total life period of a cell is generally divided into: (Hsu, 1965) 1. pre-DNA-synthetic phase (G1) 2. DNA-synthetic phase (S) 3. post-DNA-synthetic phase (G2) 4. mitotic phase (M) It was shown by Weiss (1968) that in HeLa cell cultures, protein synthesis was required throughout the S phase if DNA replication is to proceed unimpeded. Linear correla- tion was shown to exist between the relative rates of pro- tein synthesis and DNA synthesis. Seed (1965) showed that in nuclei of normal cells (monkey kidney, human skin, mouse fibroblast) DNA synthesis and protein synthesis were concurrent and were related quantitatively, but in HeLa cells there was no such correlation, probably because of continuous RNA associated synthesis of proteins. Sheek gt gt. (1960) inhibited DNA synthesis and found protein synthesis to continue on even after DNA synthesis had 24 stopped. The uptake of precursors into the RNA of non- dividing cells of rabbit macrophage cells was found to continue in the absence of DNA synthesis (Watts and Harris, 1959). In primary explanted rat heart cells, RNA and pro- tein synthesis preceded DNA synthesis and was mandatory for DNA synthesis. Weiss's observation was in agreement with the above findings (Weiss, 1968). Cessation of RNA synthesis was found to occur during cell division phase (M) but not necessarily during DNA synthesis (Feinendegen gt gt., 1960). With respect to RNA synthesis and protein synthesis, Harris (1960) did not find any mandatory coupling between the two in the rat embryo connective tissue cultures. B-Z-thionylalanine was shown to inhibit RNA synthesis with- out inhibiting protein synthesis. Seed (1965) and Stud- zinski and Lambert (1968), however, have shown that in HeLa cells RNA synthesis is concomitant with protein syn- thesis. In the above mentioned work of Harris (1960) he found 40% difference in inhibition between the nuclear and cytoplasmic RNA. This phenomenon was explained in terms of independent synthesis of nuclear and cytoplasmic RNA (Harris, 1959a). The work of Feinendegen gt gt. (1960) corroborates this observation. Tissue cultures have been used tg_ttttg for studying the genetics involved in pesticide resistance. Tamura 25 (1958) studied the embryological genetics on the resistance of different organs of different strains of Drosophila melanogaster. In his cultures at 25 ppm concentration level, he showed the existence of the difference in resis- tance to parathion (organophosphate insecticide) between strains. However, 50 ppm inhibited growth in all cultures. V. Malathion. Malathion (s—[1,2-bis-(ethoxycarbonyl)-ethy1]0,0- dimethyl phosphorodithioate) was introduced as an insecti- cide in 1950 by the American Cyanamid Company (Spiller, 1961). The structural formula is given below. Structural Formula S 0 CH 3 II II -S-CH-C-0-C2H5 CH 0 I 3 CH -c—o-c H It has a molecular weight of 330.36, melting point of 2.9°C, and boiling point of 156-157°C. It is slightly soluble in water (145 ppm) and highly soluble in various organic solvents. Decomposition is rapid above pH 7 or below pH 5. At pH 7.5 hydrolysis was 11% in 22 hours. No hydrolysis occurred for 336 hours at a pH buffered at 5.26 (Merck Index). Malathion is used in controlling various agricultur— al, forestry, stored product, veterinary, medical, and 26 general public health pests (Spiller, 1961). It is widely used in North America (Greenberg and La Ham, 1969) and is predicted to come into greater use after the expiration of its patent (Neumeyer gt gt., 1969). Part of the reason for this popularity is its low mammalian toxicity, and the vast difference in toxicity between vertebrates and in— sects (Spiller, 1961; O'Brien, 1960, 1961). There has been much evidence presented to indicate that the primary mode of action in the toxic effects of malathion is inhibition of the enzyme acetylcholinesterase. The general chain of events that occur in organophosphate insecticide poisoning has been shown to be: cholinester- ase inhibition-—»excess acetylcholine-—+nervous system destruction (by depolarization caused by acetylcholine?)—+ death (O'Brien, 1960, 1967). Some of the accompanying symptoms in mammals are convulsions, excessive parasympa- thetic activity, lacrymation and salivation (Muscarinic effects), and hyperexcitability, tremors, paralysis, and death. The inhibition is caused by the binding of the or- ganophosphates with the esteratic site of the cholines- terase in an "irreversible" manner. This combination of or- ganophosphate with cholinesterase involves not merely an ad- ditive binding but an actual phosphorylation leading to the formation of phosphorylated cholinesterase (O'Brien, 1960). 27 It has been shown that malathion is activated by de- sulfuration (oxidation) to malaoxon: 2 5 CHZ-C -0- -C2H5 CHZ-fi-o-CZHS 0 0 ISI I ll :>-SCHC0C2H—+ 3-PIS---CH-C0CH 5 CH3 This activation process occurs in various intact verte- brates and insects. The activation occurs mainly in micro- somes and the increase in anticholinesterase potency is about 10,000 fold. Thus activation is said to be an ab- solute prerequisite for toxicity (O'Brien, 1960, 1967). In vertebrates the activating enzymes are mainly found in the liver, while in insects it is found in the various parts of the body including fat bodies. Homogenization of the tissue destroys the ability to activate. Addition of nucleotides such as NAD or NADPH is said to restore the ability (O'Brien, 1967). Recently Plapp and Casida (1969) showed that the detoxifying enzymes in Mtggg were NADPH- dependent. Even though the activation reaction was brought about by oxidation, the required nucleotide for activation was reduced nucleotide (O'Brien, 1967). Although the primary factor involved in the toxicity of organophosphate compounds has been established to be the inhibition of anticholinesterase activity, malathion is suspected to have a few other factors contribute to its 28 toxicity. A possibility of another enzyme undergoing parallel inhibition has been suggested (O'Brien, 1960). The possible effect, of malathion or parts of the molecule, on various biological systems is attracting the attention of many workers. The mode of action of mala- thion in producing teratogenic effects in chick embryo has been investigated. The results showed no relationship be- tween malathion induced teratism and cholinesterase levels in the embryos (Greenberg and LaHam, 1969, 1970). In 1966 Wilson and Walker, using chick embryo cell cultures, in- vestigated such possibilities and they found malathion and mercaptosuccinate to be toxic to the cells at 3 x 10"6 M and 3 x 10"5 M, respectively. The observed result was based on cell counts. The results were the same for 95% and 99+% pure malathion. It should be noted that their cell counts took into consideration only cells attached to the bottom of the flask. The viability of the cells floating was not included. The mode of action or the probable location of induced biochemical lesions was not established. Gabliks and Friedman (1965) studied the chronic effects of malathion and several other insecticides on HeLa cell cultures. Their results showed malathion to cause 50% protein inhibition. Further studies showed re- sistance can be induced in cells against several insecti- cides. It is not clear if apparent resistance is associ- ated with selection of more resistant cells or induction 29 of enzyme(s) that detoxified malathion (Gabliks, l965a). Using insect tissue cultures derived from Antheria eucalyp- ti and Aedes aggypti, Mitsuhashi EE.El° (1970a) tested the toxicity of various insecticides including malathion. Their results also showed inhibition of growth of cells by malathion and several other compounds used. Here again. no attempt was made to find out the mode of action or the location of any biochemical lesion. MATERIALS AND METHODS I. Cell Cultures. Both insect and mammalian cell cultures were used. The insect cell cultures included one established cell line and two primary cultures, and the mammalian cultures included two established cell lines. A. Insect Cell Cultures Used. 1. Established Cell Line: Grace's Antheria eucalypti Cells. This cell line was obtained from Dr. Dean Branson (Dow Chemical Co., Midland, Michigan). Originally this cell_line was derived from ovarian tissues of Antheria eucalypti (Scott) by Grace (1962) and was adapted to Grace's insect tissue culture medium in which the hemo- lymph was replaced by fetal calf serum (7%), whole egg ultrafiltrate (10%), and bovine albumin fraction V (1%) (GMM) (Yunker gt gl,, 1967). This medium was commercially obtained from Grand Island Biological Company (Grand Island, New York; GIBCO). The cells were grown in sterile plastic bottles (250 m1 volume and 30 ml volume; Falcon 3O 31 Plastics). Initial cell concentrations of cells placed in the bottles were 40,000 to 60,000 cells. They were sub- cultured every 10 days. A fluid volume of approximately 9 ml was kept in the bottles. These standard conditions were altered as required. 2. Primary Cell Cultures: Embryonic Cells and Larval Fat Body Cells of Musca domestica L. Two DDT resistant strains (DDT-R and FC), a malathion resistant strain obtained from Dr. F. W. Plapp, Jr., Texas A & M, and a non-resistant strain of flies (NAIDM) (obtained from Michigan State Department of Agri- culture at E. Lansing) were used as the source of cells. The flies were routinely maintained on CSMA media at 30°C and 55% RH. The adults were fed 1:1 powdered sucrose and dry milk solids. The flies were routinely selected for resistance by administering DDT (1%) in food and malathion (100 ppm) in water. Embryonic cell cultures were initiated with eggs which were no more than five hours old. The eggs were first surface sterilized by washing repeatedly in dis- tilled water, and then placing them in 0.1% sodium hypo- chlorite for 20 minutes (Monroe, 1962). These were then dechorinated by placing in 3% sodium hypochlorite (Slifer, 1945) for a few seconds until most of the eggs came up to the surface. The sodium hypochlorite solution was then rapidly drawn off with a syringe and the eggs were washed 32 four times in calcium and magnesium-free phosphate buf- fered solution (CMF—PBS; Merchant gt gt., 1964). A fifth wash was given in Grace's insect tissue culture medium without hemolymph (Grace's minimal medium--GM) (GIBCO). The medium was drawn off and the eggs placed again in fresh medium (1.5 m1 packed volume of eggs/20 ml medium) and transferred to a sterile glaSs tissue grinder. The eggs were then broken up and cells dissociated with one or two gentle strokes of the plunger. Small portions of the sample were microscopically examined for optimal cell dissociation. The tissue suspension was then let stand for about ten minutes and the bottom sediment, mainly com- posed of unbroken eggs and large tissue pieces, was re- moved. The remaining tissue suspension was then trans- ferred to sterile centrifuge tubes with screw caps and centrifuged at 1000 rpm for 5 minutes. The cell-free supernatant was discarded. Cells were resuspended in Grace's minimal medium (GM) and centrifuged again and the cell-free supernatant was discarded. The cells were then suspended in 1 ml of the same medium, and 2 m1 tryp— sin (0.05%; 1:300; Nutritional Biochemicals Corporation) was added and mixed thoroughly in order to dissociate the cells further. After the cells dissociated (5-15 minutes), the cells were distributed in Grace's minimal medium sup- plemented with 7% fetal calf serum (GMF; 1/2ml/9 ml), and placed in incubators at 26°C. 33 Fat body tissues were obtained from larvae raised from surface sterilized eggs and maintained on sterile synthetic medium. The preparation of the synthetic medium was essentially the same as described by Monroe (1960, 1962). The dry medium powder was mixed with water and vitamin mixture (37g dry medium/256 m1 water/3.8 m1 vita— min solution) in a Waring blender, poured (40 m1) into milk bottles (240 ml volume), and sterilized for 15 min- utes under 15 pounds pressure at 121°C. The bottles were then removed from the sterilizer and cooled. While cooling they were constantly swirled in order to keep the medium homogeneous. Surface sterilized fly eggs, as described above, were then inoculated onto the medium (100-150 eggs per bottle) and incubated at 34°C for two days and then held at 26°C. After two days at 26°C, most of the larvae migrated to the sides of the bottle (Monroe, 1962); at this stage they were used for obtaining fat bodies. The larvae were washed in sterile water and dissected under a stereo microscope and the fat bodies collected in a Petri dish containing calcium and magnesium—free phos- phate buffered solution. The fat body cells were then washed three times by taking them through three different containers of buffer solution with the help of a wire basket or by dripping the buffer through the basket which contained the fat bodies. The final wash was given in Grace's minimal medium and placed in modified Grace's 34 medium. About 100 fat body pieces, each containing about 200 cells, were placed in 3 ml medium and incubated at 26°C and used as needed. B. Mammalian Cell Cultures. Chinese hamster cells (quasi-diploid epithelial cells, CH-461) and normal human amnion cells (AV3) were obtained from Dr. J. Trosko (Michigan State University) and maintained in our laboratory for our experimental pur- poses. The Chinese hamster cell line was originally provided by Chu (1965) who obtained it from Yerganian (1958). The generation time of the cells was approximately 24 hours. They were grown in minimum essential medium (MEM) (Eagle; with Hank's salts) and supplemented with 10% fetal calf serum (GIBCO) in plastic tissue culture bottles. The pH was adjusted with sodium bicarbonate (0.35 g/l; GIBCO). Subculturing was made when the floor of the bottle was completely covered with cells. The normal human amnion cell line was originally ob— tained from American Type Culture Collection, Rockville, Maryland. The generation time of the cells was approxi- mately 24 hours. They were grown the same way as the Chinese hamster cells. 35 C. General Sterile Procedure. All cell culture media and solutions used for washing cells were provided with antibiotic stock solution (penicillin 10,000 units/m1 + streptomycin 10,000 g/ml) and anti-pplo (100x; GIBCO) each 1 m1/100 ml of the solu— tion. Germ-free working conditions were provided in a sterile laminar flow hood. Necessary equipment and glass- ware were sterilized by autoclaving for 30 minutes at 121%; Liquids were sterilized only for 15 minutes. Solutions that could not be autoclaved were sterilized by using "Steitz" Bacterial Filter (Hercules Filter Corporation). Sterile pipettes (disposable; 10 ml, plastic pipettes Falcon Plastics and 1 ml glass pipettes, Corning Glassware) and sterile syringes (plastic, disposable; B-D) were used in transferring cells and liquids. Cells were exclusively transferred through glass pipettes. D. General Microscopic Observations. Observations on cell cultures were made through the inverted microscope (Wild M 40). When needed the re- search microscope (Wild M 20) was used. 36 II. Studies on the Interaction of Malathion and Malaoxon with Cell Cultures. A. Pesticide Samples Used. Non-radioactive malathion samples include tech- nical grade (95%), secondary grade (97%; American Cyana- mide Co.), analytical grade (99.7%; American Cyanamide Co.), and further purified samples. 14 Radioactive malathion samples include C - labeled (-diethy1 [maleate-2,3-14C]; 98% pure; Nuclear-Chicago Corp.), 35 and 32P - (Amersham-Searle Co.) compounds. S — (both atoms labeled; Amersham—Searle Co.), Malaoxon sample was obtained from American Cyanamide Co. (analytical grade) and further purified as needed. B. Malathion and Malaoxon Purification Procedures. Malathion and malaoxon samples were dissolved in acetone, streaked on thin layer chromatography plates (TLC; Silica Gel G; 250 u thick; Analtec Inc.), and then developed. The compounds were identified with the help of a co-chromatogrammed sample sprayed with chromogenic reagent and the colors of the compounds developed by heat- ing the plates 110°C for 2-3 minutes. The developing solvents were: 37 1. Chloroform - cylohexane — acetone 10 : 10 : 1.2 2. Hexane - 1,1,1 trichloroethane — methanol 10 : 3 : 0.5 3. Hexane - 1,1,1 trichloroethane - methanol 50 : 15 : 0.5 The different bands were scraped off and the compounds eluted with acetone. Samples were checked for purity by re-chromatographing small portions. The samples were cleaned repeatedly until a single spot was obtained for the desired compounds. These samples were dried under nitrogen, weighed, and made up to desired concentration in acetone. The chromogenic reagent used was 0.5% solution of 2,6-Dibromoquinone-N-chloro-p-quinoneimine (= 2,6-dibromo- quinone chloroimide) in cyclohexane (O'Brien, 1960). C. Malathion and Malaoxon Inoculation Procedure. Malathion and malaoxon were dissolved in acetone and inoculated into the cultures. The volume of acetone per 9 ml of culture media never exceeded 18111. While inoculating, care was exercised to see that the solution (acetone + pesticide) first mixed with the medium before coming into contact with the cells. The overall dose range of pesticides for the entire work was 0-150 ppm. 38 D. Malathion and Malaoxon Extraction Procedures. Malathion and malaoxon extraction procedure described by Matsumura and Brown (1961) was slightly modi- fied for our purpose. The cells together with the media was sonicated at power 20, tune 2 (Blackstone Ultrasonic Inc.). The pH was then brought to neutral (pH 7.0) by using potassium hydroxide (1 N) and hydrochloric acid (1 N). Double distilled water (pH 7.0) was added to the homogenate (11 ml water/9 ml homogenate) and extracted with large volumes of chloroform (100 m1 chloroform/20 m1 sample). Such large amounts of chloroform was necessary to avoid emulsion formation. The sample was thus extracted three times. The pooled chloroform was then washed with double distilled water (pH 7.0), vacuum dried, and sus— pended in convenient amounts of clean hexane for gas-liquid chromatographic and thin layer chromatographic analysis. The chloroform fraction contained malathion and malaoxon and the water fraction contained most of the degraded malathion products (Matsumura and Brown, 1961). The re— covery efficiency of this procedure was 90%-95%. E. Malathion and Malaoxon Identification and Quantification Procedures. Identification was done by TLC (as described earlier), gas-liquid chromatography (GLC; Packard Instru- ment Co.), and radio isotope methods. Both phosphorus 39 detector (alkali flame ionization detector) and electron capture detector were used. Gas-liquid chromatographic analysis parameters: Column: Glass, 3.3' x 1/4" Packing: Diethylene glycol succinate (Applied Science Laboratories Inc.) 2% (w/w) on 100/200 mesh Gas ChromrQ (Applied Science Laboratories Inc.) (Corley and Beroza, 1968) Carrier Gas: Nitrogen 40 cc/min. Other Gases: (for phosphorus detectors only) Air’ 150 cc/min., hydrogen 5 cc/min. Temperature: Inlet 190°C, Outlet 190°C Detector 190°C, Column 180°C Temperature parameters were decided upon after making sure, with a help of mass spectrometer (LKB Gas chromatogram mass _spectrometer, Model 9000), that the samples did not undergo any significant change at these temperatures. Column was conditioned for two days before use. Experimental samples were injected after getting consistent responses for 4 ng malathion and 40 ng malaoxon standards. Quantitation was generally done by measuring the area (1/2 hb) of peaks and comparing with those of the stand- ards. 40 Conversion of malathion-14C to malaoxon was checked by collecting the effluents corresponding to the peaks obtained and determining the radioactivity in the frac- tions. The effluents were collected on glass wool (Pyrex) packed in glass cartridges. A Packard Model 850 gas fraction collector was used for this purpose. Collector temperature was 190°C. F. Radio Assay of Metabolic Precursors Used. Radioactive thymidine (methyl-3H; 98% pure; 2 Ci/mM, 0.25 mCi/0.5 m1; Amersham-Searle Co.) was made up to 5 ml in sterile water to give a final concentration 7 mM/Ul (6 x 10.1 Ci/ul) and stocked for use. of 2.5 x 10‘ Three to 4 pl of this stock was inoculated per 9 m1 of media. The concentration of uracil-3H (0.5 mCi/6.25 mg, 89 mCi/mM; Cal-Biochem) stock solution was 0.5 mc/ml sterile water. Two to 3 ul of the solution per 7 to 9 m1 of the media was used. Phenylalanine-14C (in 1.0 N HCl solution, 0.5 mCi/mM; Nuclear-Chicago Corp.) was diluted in sterile water to give a final concentration of 1p Ci/ul and used in our experiments. Radioactivity of samples was determined by using a liquid scintilation counter (Mark I; Nuclear-Chicago Corp.). Omnifluor (98% PPO + 2% Bis-MSB) (New England Nuclear Corp.) solvent system was used for non-aqueous samples; for aqueous samples omnifluor solvent and Triton‘G> 41 X-lOO (Packard) mixture (2:1) was used. The aqueous fraction for the latter was maintained at 23%-50% level. The incorporation of thymidine-3H and uracil-3H into DNA and RNA were determined in the following manner. The cell cultures were collected in centrifuge tubes, centri- fuged (1700 rpm; 8 min.) and the cells separated. In the case of Chinese hamster and human amnion cell cultures, the cells were first removed from the bottom of the flask with a rubber policeman. They were then washed in phos- phate buffered solution (GIBCO) three times through re- peated suspension and centrifugation. The method used for DNA synthesis was basically the same as described by Bollum (1959) and modified by Regan and Chu (1966). The cell pellets obtained after the last centrifugation were resuspended in 5 ml buffer solution (PBS; GIBCO) and sonicated at power 20, tune 2 for 30 seconds. One hundred to 200 ml aliquots were placed on Whatman 3 mm paper disks (W & R Balston, LTD.), washed in cold TCA (10%) three times (15 minutes each the first two times and the third time left in the refrigerator over night), then transfer- red to absolute ethanol (10 minutes) then to acetone (10 minutes) and then taken out and dried. The dried disks were placed in scintilation vials and the radioactivity was counted. The same method was used in the case of uracil-3H. Uptake values were obtained by placing 100-200 42 U1 aliquots on the disks and counting the radioactivity after they were dry. They were not washed in TCA, ethanol, or acetone. The radioactivity counted on the disks was entered as percent incorporation or percent uptake in terms of the total amount inoculated into the media. In the phenylalanine-l4C uptake studies, the labeled amino acid was directly added to the medium (0.5 to lu 1 per ml of the medium). After the incubation period, cells were separated from the medium, washed, and were sonicated. Specific amounts of the homogenate were placed on Whatman 3 mm paper disks, dried and counted. Percent uptake in relation to the total amount of labeled compound placed in the culture were determined and recorded. 1 The amount of incorporation of the labeled pre— cursors were calculated for unit amounts of protein (dpm/ tlg protein). G. Determination of Protein Content. Protein content of cell homogenate samples were determined by Lowry's method (Lowry gt gt., 1951) and by taking weights of aliquots dried on pre-weighed Whatman 3 mm paper disks. The cell culture samples whose protein content had to be determined were sonicated, brought to pH 7.0 and made up to required volume in water (double distilled, 43 pH 7.0). In the case of homogenates of cells separated from the medium, 4 x 105 to 4 x 106 cells were made up to 5 m1. In the case of cells plus media, 9 ml of the homo— genate was made up to 20 m1. A series of standards were set up by using albumin crystals. H. Mitotic Inhibition Studies. Malathion and malaoxon effects on cell repro- duction were also determined by the extent of inhibition of cells entering the mitotic phase. This was estimated by counting the number of cells which had entered meta- phase (Figures 13 and 14). This study was made only on Chinese hamster cell cultures. The cells from full-grown cultures (30 ml volume ’ bottle) were trypsinized (3-1/2 ml/bottle), 1.5 ml samples of the pooled cell suspension were placed in new bottles, and 7.5 ml medium was added to them. The cells were in- cubated until about one-third of the surface area of the bottom of the flasks were covered with equally distributed attached cells. The medium was then replaced with fresh medium and desired amounts of pesticides under study were added. To each bottle 1 ml sterile Colcemide (10 (lg/ml isotonic saline solution) per 9 m1 of the medium was added and incubated for 5 hours. The medium was then poured off. Warm (37°C) 1% sodium citrate solution was then added to the cultures and set aside for 1 hour. The solu- tion was poured off and 5 ml of freshly prepared Carnoy 44 fixative (1 part glacial acetic acid : 3 parts reagent grade methanol) was added. The fixative was changed three times. The first change after 3 minutes, the second after 5 minutes, the third after 10 minutes. After the third change, the cultures were placed in a refrigerator over night. After the fixative was decanted, the bottoms of the flasks were then cut out with a hot blade and the edges leveled and smoothed. The "slide" was then dipped in 70% methanol (the excess shaken off) and quickly passed over the flame and dried. A few drops of 1% aceto-orcein (Merchant gt gt., 1964) was placed on the slides and a cover slip was placed on top. Care was taken to see that the aceto-orcein did not dry. After the chromosomes had taken up the dye, excess aceto-orcein was removed. A drOp of glycerine was then placed on the cells and a cover slip was placed on tOp. The slides were made semi-permanent by sealing the edges with nail polish. In later mitotic inhibitory studies Lab-Tek® tissue culture chamber/slides (Miles Laboratories Inc.) were used. The procedure was basically the same as described earlier except that the trypsinized cells were first mixed with culture medium (1.5 ml cell suspension/7.5 ml culture Vmedium) and then 0.2 m1 samples of the suspensions were placed in each chamber. After the cells had attached to the bottom (8 hours), 0.02 ml Colcemide and desired amounts of pesticides were added to the chambers. The 45 amount of fixative and hypotonic solutions used were 0.5 ml each. The advantages of using these chambers over regular culture bottles were many. This method required less solution, provided more replicates (8 chambers/slide), and provided a regular glass slide for observation. I. Autoradiography of TLC Plates. The plates carrying labeled compounds were auto- radiographed when needed. Kodak medical x-ray films were exposed to plates for 20 days and developed. The spots were compared with the colored spots obtained on the plates after spraying them with specific chromogenic reagents. J. General Studies on Fat Body Cells. Fat bodies were tested for a few enzymes, DNA synthesis, and protein synthesis in order to get an under- standing of their metabolic state in £g_ytttg tissue cul- ture conditions. Histochemical tests were performed for the enzymes. DNA synthesis was assayed by stripping film autoradiography and estimating thymidine—3H incorporation using cell homogenates. 1. Histochemical Tests. Fat bodies incubated in modified Grace's medium for four days were washed in Grace's minimal medium, placed on cover slips, briefly frozen, and placed in in- cubation medium. Assays were performed for glutamic de- hydrogenase, ioscitric dehydrogenase, o-glycerophosphate 46 dehydrogenase, malate dehydrogenase, lactate dehydrogenase, and B-hydroxybuteryl dehydrogenase. The methods used were according to Pearse (1961). The controls consisted of in- cubation mixture minus substrate. 2. Stripping Film Autoradiography. Thymidine-BH was used in the medium (3111 stock). The fat bodies were assayed for thymidine incor- poration into DNA by the Stripping Film Autoradiographic Method and TCA precipitation of cell homogenates. The latter is described previously. The stripping film technique was basically the same as described by Schmid (1965). The fat body mono-layer cells were placed on gelatinized (2.5 g gelatin/500 ml water) slides and a cover slip placed on top. The auto- radiographic films (AR-10; fine grain; Kodak) were cut to desired size and mounted on slides in the darkroom. Expo- sure period was 4 days. The cells were stained with Giemsa stain. III. DDT Metabolism in Musca domestica Embryonic Cell Cultures. The procedure for setting up primary embryonic cell cultures was described previously (p. 31). The cultures were treated with 1.0 ppm DDT. Acetone and dimethyl sul- foxide were used as carrier solutions for DDT. Cultures were incubated for 3 days at 26°C. At the end of the 47 incubation period, cells and the media were extracted. The extract was analyzed through gas-liquid chromatography for conversion of DDT——+DDE. Gas-liquid chromatographic analysis parameters: Column: Glass 6' x 1/4" Packing: 10% DC-200 Carrier Gas: Nitrogen 170 ml/min. Detector: Nickel foil-electron capture Temperature: Inlet 220°C, Outlet 200°C Detector 210°C, Column 190°C Relative quantitations were made by measuring and comparing the areas of the peaks. The extraction method of DDT and its metabolites was basically the same that was used by Oppenoorth and Voerman (1965). Extraction samples were not treated with Nast4 solution. One ml of the extraction solvent (2 part cyclo— hexane : 1 part isopropanol) was used every 1 ug of DDT inoculated in our culture system (8 ug/Bml). Three ul final solution (cyclohexane) was injected and peaks measured. IV. General. The values obtained for the different experiments were based on two or more replicates. RESULTS 1. Antheria eucalypti Cell Cultures. Standard growth curve of the cell line and standard DNA synthesis was first studied and the results are given in Figures 1 and 2. A. Growth Curve and the Estimated Doubling Time. (Figure l) The starting cell number was z 80,000 cells/ml. The initial cell concentration was established by using a Hemocytometer (Fisher - E. & A.). Subculturing was done by splitting the cultures after the third day and adding new medium (4 ml culture + 4 m1 new medium). The doubling time calculated during the log phase was 4.16 days. In general a straight line increase in number of cells was noticed up to 8 days, except for a slight lag during the first day. B. DNA Incorporation of Tritiated-thymidine. (Figure 2) Incorporation of thymidine-3H was monitored for 9 days. There was a lag period during the fourth and the fifth days. 48 Number of Cells/Unit Area 49 Time in Days ult- -. -r —1)- .1- qt- .4- ‘\ T . 26-. 24:- o + t + : 40 80 120 160 Time in Hours Fig. 1. Growth curve of Grace's Antheria eucalypti established cell line. 50 T. 10'? .,..¢ "' 'O 0 z ‘- d) I: 4’ a. E 2 “I. ah- 8 .,.' 5" 4.) 16 H ‘- O O: H 0 db 0 G H w ‘1- 0 t : : : .L : 1 t : 5 Time in Days '31 Fig. 2. Incorporation of thymidine-3H into DNA by Antheria eucalypti cells. Thymidine-3H incorporation was determined for unit wet weights of cell homogenates. The percent incorporation was determined in relation to the total activity placed in the medium. :12 uCi/ZO m1) 51 C. Effect of Malathion on DNA Incorporation. (Table 1) The study was conducted to evaluate the time/ effect correlation for the insecticide on DNA synthesis of the cell line. The dosage of malathion tested was 75 ppm. This test dosage was arrived at by microscopically evaluating the effect of different doses (0, 50, 75, 150 ppm) on the cells. At 75 ppm approximately 50% of the cells showed signs of cytotoxic effects, which are given elsewhere, after 4 days. Thymidine-3H incorporation into the medium, homogenate, and TCA precipitated homogenate TABLE 1. Effect of Malathion on the Incorporation of Thymidine- H Uptake by Antheria eucalypti Cells. Culture Conc. of Time - Time Malathion Thymidine- H % éfiggiggizffign 0f (Hrs.) (ppm) added 4 0 1 hr not significant 75 1 hr not significant 72 0 1 hr 100 75 1 hr 55 75 24 hrs 59 96 0 1 hr 100 75 1 hr 67 150a 1 hr extensive cell death a At this concentration most cells were dead and extensive deggneration symptoms were noticed, therefore, no thymidine- H incorporation was determined. 52 was determined. Thymidine incorporation was estimated in relation to unit protein amount of cells (Lowry's method). The nucleotide incorporation in the control (0 ppm) sample was used as 100 percent. D. Effect of Malathion (75 ppm) on Protein Syn- thesis. (Table 2) Protein synthesis was analyzed after 15 days incubation, on the basis of uracil—3H incorporation into RNA and final protein content. Uracil-3H incorporation was determined for ugs of protein. The protein content was determined by Lowry's method. The values obtained for control samples (0 ppm) were assumed to be 100% and the rest evaluated accordingly. TABLE 2. Effect of Malathion (75 ppm) on the Protein Synthesis of Antheria eucalypti Cells. Conc. of Uracil-3H Malathion Incorporation Protein Content (%) (PPm) into RNA (%) 0 100 t 8.7 100 i 2.2 75 nil 28.7 i o 53 E. Cytological Effects. (Figures 3, 4, 5, 6; Table 3) The effects were noticeable at dosages above 75 ppm and after 4 days. The effects observed were round- ing up of cells, enlargement of cells, and high granula- tion. Feulgen test (Merchant gt gt., 1964) showed the DNA to be scattered over the cells irregularly. In the healthy cells the Feulgen positive areas were confined to definite nuclear areas. F. Selection for Malathion Resistance. By selection Antheria eucalypti cell cultures with resistance up to 225 ppm have been obtained. TABLE 3. Malathion Effect on the Viability of Antheria eucalypti Cells Based on Morphological Aspects. Conc. of Hours Malathion (ppm) 24 48 72 96 0 (Control) ++++ ++++ ++++ ++++ 50 ++++ ++++ ++++ -:¥+ 75 ++++ ++++ ++++ --++ 125 ++++ -+++ --++ --—+ Note: take up trypan blue are indicated by "+". are indicated by "-". cells. Normal healthy looking cells which did not Small, rounded cells with extensive granulation which took up trypan blue The latter cells were degenerate Fig. 3. Antheria eucalypti cells. "Normal" culture. Magnification 150x. Polari21ng filter (33mm) used. No stain. Fig. 4. Antheria eucalypti cells. Telophase cells shown. Magnification 150x. Feulgen stain. _._._ .. .- Fig. 5. Antheria eucalypti cells. Anaphase and prophase cells shown. Magnification 600x. Feulgen stain. Fig. 6. Antheria eucal ti cells. Malathion treated (75 ppm). S-da -old cuIture. Magnification 600x. Feulgen stain. 56 II. Studies on Embryonic Cells of Musca domestica. (Figures 7 and 8) A. Culturing of Embryonic Cells. Grace's minimal medium supplemented with 7% fetal calf serum (GMF) was found to be the most suitable medium for culturing the cells. Tissue Culture Media 199 (TC Medium 199; GIBCO), minimum essential medium (MEM; Eagles +; +10% fetal calf serum; GIBCO), and modified Grace's medium (GMM) were found to be not too effective. In TC 199 and MEM the cells showed high granulation and subsequent degeneration within one week. There was no noticeable attachment of cells to the bottom of the flask. In GMM there were a few attached cells. A few of the cells were viable for up to 15 to 20 days, but granulation, vacuolation, and swelling of the cells were pronounced. In GMF, which was our modification of Grace's minimal medium (GM), a high number of cells were viable for more than 20 days. In GMF two prominent types of cells were noticed: l. rounded cells which were found floating, and 2. elongated cells which attached to the bottom of the flask, most of which showed protoplasmic connections to the neighboring cells. (Figure 8) Many of the protOplasmic processes showed contractile movement which lasted for more than 20 days. During the first week the contractions were observed to be irregular, Fig. 7. Musca domestica embryonic cells. 1 hour old culture. Cells unattached. Magnification 150x. Polarizing filter (33 mm) used. No stain. Fig. 8. Musca domestica embryonic cells. 8-10 hours old culture. Cells attached to the bottom. Magni- fication 300x. Polarizing filter (33 mm) used. No stain. 58 but during the second week a few rhythmic contractions were noticed. Both the attached cells and the rounded floating cells were found to increase in numbers, extensively during the first week, and then slower growth. Significant gran— ulation appeared only after 25 or 30 days. Seven percent addition was found to be more effective than the 10%, and was used in subsequent experimentation. Attempts were made to subculture the attached cells from crowded cultures by trypsinizing and re-suspending in fresh medium. So far we have not successfully subcultured these cells. The subcultured cells did not attach to the bottom, showed granulation and disintegration within a week. Only a very few cells were found viable but floating after seven days. B. The Effect of Malathion on the Embryonic Cell. DNA synthetic activity in untreated cultures was demonstrated up to a period of 8 days (which was the end of the test period). The cells were incubated at 26°C for 4 days and treated with thymidine-3H and in- cubated for another 4 days. After which they were tested for thymidine-3H incorporation. Cells were homogenized and 100 pl aliquots were assayed for thymidine-3H incor- poration. The studies indicated active DNA synthesis by the primary house fly embryonic cells. Effects of mala- thion on embryonic cell cultures from malathion resistant flies (M-R) and from non-resistant flies (N-R) were also 59 studied. Data was obtained up to 100 ppm malathion treat- ment on M-R cultures, but due to contamination of the N-R cultures, the analysis of these cultures was not completed beyond 30 ppm (Figure 9). Analytical grade malathion (99.7%) was used in these experiments. Readings were taken for 10, 30, and 50 ppm malathion dosage on 4-day, 6-day, and 8-day-old cultures, respec- tively, and compared with controls. Controls did not contain malathion. There was apparently no effect of malathion on the contractions of protoplasmic processes up to 50 ppm in the embryonic cell cultures of non- resistant flies. III. Studies on Fat Body Cells of Musca domestica Larvae. Fat body cells were obtained from the different strains of house flies and incubated in modified Grace's medium (GMM) and thymidine-3H incorporation was studied. The TCA precipitated cell homogenate ("TCA soluble frac- tion") did not show any significant activity. The auto- radiograms of cells also gave negative results (Figure 23, Appendix). In phenylalanine-14C treated cultures, the fat body homogenate showed significant uptake. Fat bodies were assayed for enzyme activity with re- gard to a few selected enzymes (Table 4). Corresponding figures are given in the Appendix (Figures 23, 25, 26, Appendix). 6O ZOO-- Thymidine-3H % Incorporation r I 1 I 0 iiiiiillifl 20 4o 60 80 100 ppm Malathion Fig. 9. Effect of malathion on thymidine-3H incor— poration in embryonic cell cultures of Musca domestica. One day old cultures were used in these experiments. The treated cultures were incubated for 3 days. 61 TABLE 4. Histochemical Studies on 4-day—old Fat Body Cultures of Musca domestica Larvae. NO With Enzymes Substrate (Control) Substrate Glutamic dehydrogenase - + Isocitric dehydrogenase - + -glycerophosphate dehydrogenase - + Malate dehydrogenase - Lactate dehydrogenase - -hydroxy buteryl dehydrogenase - - IV. Studies on Human Amnion Cells. The effect of malathion (25 ppm, 99.7% pure) on the incorporation of thymidine, uracil, and phenylalanine were studied (Figure 10). In this experiment after the incubation period of 30 hours, the medium was poured out and the remaining cells in the bottle (attached to the bottom) were rinsed and the cells were then trypsinized and removed for analy- sis. Determination of phenylalanine uptake was studied by placing aliquots on disks and counting them in the scintillation counter. The viability of the cells was determined by esti- mating the protein content of the cells that remained attached to the bottom of the flask. 62 100? 90* U 80; 70db 3 Uracil- H 60‘:- Phenylalanine-14C 40‘ % Incorporation U1 0 i 301 29' .. 3 Thymidine- H 10- I l l l l n 0 . I v I . 5 10 15 20 25 30 Incubation Time in Hours Fig. 10. The effect of malathion (25 ppm) on the incorporation of thymidine, uracil, and phenylalanine in human amnion cells. The radioactive precursor incorpora- tions and uptake were related to the unit amounts of cellular protein. (Controls = 100%; protein corrected for trypsin). Reduction in the viability of malathion treated cultures or reduction of total cell protein was found to be 42%. 63 V. Studies on Chinese Hamster Cells. A. Inhibition of thymidine-3H Incorporation by Malathion. Effect of different dosages of malathion (99.7%) on the incorporation of thymidine-3H was evaluated. The values obtained for 0 ppm, 20 ppm, 40 ppm, and 50 ppm concentrations of the pesticide for a 30-hour incubation time are reported in Figure 11. The 6-hour study is re— ported in Figure 12. The study was made on the cells that remained attached to the bottom of the culture flask. These cells were scraped off and not trypsinized. The cells were collected by centrifuging at 1700 rpm for 10 minutes. The cultures treated with 40 and 50 ppm mala— thion had relatively fewer cells attached to the bottom after 30 hours, but enough to determine the protein con- tent of the cells. B. Inhibition of Mitosis by Malathion. (Figure 13) The effect of malathion (99.7%) at 25 ppm and 40 ppm concentration was a reduction of mitosis of 50% and 85%, respectively. Colcemide was used to arrest the cul- tures at metaphase and the mitotic cells counted included cells that were in prophase through metaphase (cells with well-defined chromosomes; Figure 14). There were two dif- ferent studies. The incubation time was 5 hours for the first and 10 hours for the second study. The extended 64 100;- c O «- -H 4.) m ’2". d- u 8 «- 5 u 20 ppm m "H m 50" c -a '0 ,E‘ 4.- 40 .c PPm [-4 j“ m- 50 ppm 0 4. 4. 15 30 Incubation Time in Hours Fig. 11. Effects of different dosages of malathion on the thymidine- H incorporation in Chinese hamster cells. Thymidine-3H incorporation was determined for unit amounts of cellular protein. (Control = 100%) 65 120'" 100« 80- 60‘ 40. 0 ppm 1 ppm 25 ppm 50 ppm % Thymidine-3H Incorporation 20- Fig. 123 Effect of malathion on the incorporation of thymidine- H by Chinese hamster cells during the first 6 hours of incubation. Analytical grade (99.7% pure) malathion was used. 66 .mmHmEMm topmouu coficumaofi now monsoon mums mHHoo Am.ma av mmm paw .mpflam Houucoo some now pmucsoo mum3 maaoo Am.HH «V HNHH mo ommuo>¢ .Hmofimso mom ooucooo mums muomm 039 unmoa Dd .c0fiumuucoocoo some now poms mum3 Aoowam \mumofimco we mmowam 039 .muoon oa mp3 ucmsummuu nmumm pofluom cowumnsocH .poflosum mo3 cownuoams 8mm ow mo uommmm .poms mums moowam\uon5mno ououaso oommfluAmwoalnmq .HH monum .AcoflnumHmE ocv Houucoo opp £DH3 poumqeoo mm3 cowcumame Sam on mo uommmm .maaoo owuoufle m mm pmmmmumxm ma ucmfloflwmooo caucus: .maamo Hum mo Hmuou o How mmpflam omummuu cownpoamfi ca pousooo mums muomm cm>mm new xam .mHHmo now no Hmuou o How mowam Honucoo comm cw poucsoo mums muomm Hsom .muson m mm3 mpflfimoaoo pom coanumamfi mo ucmEpmmHu may nouns moaned cosponsocH .ooms mums Acofipmuucoocoo mom my mmauuoo musuaoo comma» ofiummam .H upsum .mmusuaso Hamo Hmumfimo ommcwso ca soHnumHmE an mHmouHE mo GOADHQHSGH .ma .mwm cownumHmz 8mm coagumamz and as o 0 cm 0 o tn :m 11¢ % 11¢ % 11$ m iro O a... 3. I. r. 5m 3 gym 0 D 3 3 3 1: n :3 n s S .aH .-~H Lea ..¢H HH ansum H spasm 67 Fig. 14. Chinese hamster cells. "Normal" culture. Treated with Colcemide. Magnification 300x. Aceto-orcein stain. 68 incubation was used to allow more cells to enter mitosis. The mitotic coefficient is expressed in terms of percent mitotic cells per total cells counted. C. General Cytological Effects. Microscopic observations were also made on the morphology of cells in 20 ppm, 30 ppm, and 40 ppm cul- tures. Extensive vacuolation was noticed in those cul- tures. Vacuoles, though noticed in untreated cultures, were not so extensive (Figures 15, 16, 17, and 18). Fur- ther, in 30-80 ppm malathion treated cultures, 12-18 hours after treatment, the cells rounded up and detached from the surface. This phenomenon seemed to be directly proportional to the concentration of malathion placed in the cultures. At 80 ppm no cells were attached to the bottom of the flask after 24 hours, while at lower concen- trations (20, 30, and 40 ppm) lesser numbers of cells underwent rounding up and floating. No precise quantita- tion of this phenomenon, based on cell numbers, was made. Another correlated phenomenon observed was that the medium in which the cells were degenerating turned alkaline as detected by the change of color (orange to bluish-red) of the phenol red indicator in the medium. Eighteen to 20 hours after the treatment, the alkalinity was noticed to be directly preportional to the concentration of malathion at 20-80 ppm range. However, no precise quantitation was made. No color change was noticed in the medium which had 69 Fig. 15. Chinese hamster cells. "Normal" cultures. 10 hours old cultures. Phase contrast. Magnification 300x. Polarizing filter (33 mm) used. 70 Fig. 16. Chinese hamster cells. Malathion treated (30 ppm). 24 hours after treatment. Extensive vacuola— tion. Phase contrast. Magnification 300x. Polarizing filter (33 mm) used. 71 Fig. 17. Chinese hamster cells. Malathion treated (50 ppm). 10-12 hours after treatment. Magnification 150x. Polarizing filter (33 mm) used. No stain. Fig. 18. Chinese hamster cells. Malathion treated (50 ppm). 24 hours after treatment. Cells attached to bottom. Magnification 150x. Polarizing filter (33 mm) used. Aceto-orcein stain. 72 malathion but no cells. Medium with phenol red is reddish-orange at pH 7.0, yellow at pH 6.7 and below, and bluish-red above pH 7.5. D. Effect of Malathion and Malaoxon on the Multi— plication of Chinese Hamster Cells. Malaoxon had been shown to be many times more toxic to animals than malathion and the toxicity of mala- thion is mainly due to the conversion of malathion to malaoxon (O'Brien, 1967). This study was one of the studies conducted to evaluate the effect of malaoxon on tissue cultures. The effect of malaoxon was evaluated with regard to its effect on cell multiplication and compared with that of malathion at 20 ppm dosage (Figure 19). The results show that malathion is more effective in inhibiting cell multiplication than malaoxon at that dosage level. The effect of malathion at 20 ppm and 40 ppm dosage levels indicate that at 20 ppm there was an increase in cell number over the initial concentration during the 24 hour period. The increase was 75.2% of the increase noticed in the controls. At 40 ppm after the end of 24 hours, the number of viable cells was lower than the ini- tial concentration. The effect of malaoxon at 5 ppm and 20 ppm indicated that at 5 ppm there was no significant decrease in multi- plication as compared to the controls. The difference in 73 120«- . Control Malaoxon 5 ppm . Malaoxon 20 ppm 100-- 3 - Malathion 20 ppm p 80.L -a s a \. u m g 60* 2 H . a . Malathion 40 ppm 0 40¢- 20-L 1 0 2'4 Incubation Time in Hours Fig. 19. Effect of malathion and malaoxon on the multiplication of Chinese hamster cells. The inner rec- tangle of the eyepiece was used as the unit area in deter- mining the cell number. Magnification of 300x was used. Duplicate samples were set up and 5 unit areas per sample were counted and averages determined. The initial cell count for all the samples was 62 per unit area. Average cell number per unit area was used in the graph. SE for cell counts = 9.6%. 74 number of cells between the 5 ppm malaoxon treated cul- tures and the controls after the 24-hour incubation period was less than 5%. However, at 20 ppm the differ- ence in final concentration of cells was 14.9%. The malaoxon sample used here was a highly purified sample (100%). The unpurified sample (analytical grade) destroyed almost all the cells at 1 ppm! There were no attached cells in the cultures even after 20 hours. No further quantitative "dose-mortality" tests were done with the non-purified malaoxon sample. At 1-20 ppm malaoxon ("100% pure") did not show any significant inhibition of thymidine-3H incorporation by Chinese hamster cells (Figure 20). VI. Comparative Study on the Effect of Malathion, Malaoxon, and "Impurities" Found in Malathion and Malaoxon Compounds. (Table 5) The disparity noticed in the effect of unpurified malaoxon and purified malaoxon (See Section V. D.) ini- tiated studies to find out the "impurity(ies)" that was/ were responsible for the toxic effect noticed with the unpurified malaoxon sample. This study was further ex- tended to find out if the toxicity observed with malathion was also due to impurities found in the sample. In the above mentioned solvent systems good separation of mala- thion and Mx4 can be obtained only if the concentration of 75 1401- 1 ppm 120-- 5 pmeO ppm 8 2.5 ppm '43 100.» - Control u o O: H 8 c 80-- H m m l 8 .H 60‘»- 'U E. .G B / w 40-- ” 20" 0 i Incubation Time in Hours Fig. 20. Effect of malaoxon (0-20 ppm; 100% pure sample) on the thymidine-3H incorporation by Chinese hamster cells. Control had no malaoxon. The incorpora- tion in the control cultures was assumed to be 100% and the rest determined accordingly. 76 TABLE 5. Malaoxon "Impurities" Separation. Major Compounds Detected Rf Values Malaoxon "6" (Mx6) 0.64 Malathion 0.57 Malaoxon "4" (Mx4) 0.49 Malaoxon 0.57 Malaoxon "0" 0.07 Note: Silica Gel G (Analtec Inc.) 25011 plates were used. Solvent system used for developing: hexane - 1,1,1 trichloroethane - methanol 10 : 3 : 0.5 Developing time was 2 hours. The front = 14 cm. the compound is not more than 1119. For that reason the above solvent system was not used for TLC purification of those two components of the impure sample. Separation of malathion and Mx4 from the crude malaoxon sample was best obtained with the following developing solvent: Hexane - 1,1,1 Trichloroethane - Methanol 50 : 15 : 0.5 TLC purification of malathion was performed with the use of this solvent. TLC analysis of secondary grade (97%) and analytical grade (99.7%) malathion showed a yellow spot with a R value corresponding to that of Mx4 found f in the malaoxon sample. That particular "impurity" had not been tested on cells due to the difficulty in obtain- ing enough material to test. However, a comparison be- tween the "impure" malathion and the malathion sample 77 purified in our laboratory (presumed 100%) did not differ greatly in their effect produced on cells with regard to inhibition of thymidine-3H incorporation. The purified compounds (presumed 100%) from the crude malaoxon sample were tested for their relative ef- fects on thymidine-3H incorporation of Chinese hamster cells. Mx II was obtained with the following solvent system: Chloroform — Cyclohexane - Acetone 10 : 10 : 1.2 The Rf values were 0.75 for malaoxon and 0.66 for Mx II (silica Gel G; Analtec Inc.; 250). Mx II was the lower half of the malaoxon spot. Since it trailed behind mala- oxon and was more yellowish in color, it was decided to ,test that fraction also. It was evident only in the above mentioned solvent system. Twenty ppm dosages were used. Results are given in Figure 21. Mx4 was found to be the most toxic of the major compounds purified from the malaoxon. Attempt was made to identify the Mx4 compound through mass spectrometer. The compound so far has eluded identification. Analysis indicated the mass of the compound to be 292. 78 120;- l “100..”- : o -a 4.) g Mx (Malaoxon 3‘ 80.. o o g Contro m m! 4. a) 60 s ;a 'o -a E Malathion ,1: 40-h B .- 00 D M x 0 F 20.1 M x 4 0 4% 30 Fig. 21. Effect of malathion (purified 100%), malaoxon (purified 100%), and "impurities" on % thymidine- H incorporation by Chinese hamster cells. Dosage used = 20 ppm (each). Lowry's method was used in determining the protein content. Controls had no pesticide. 79 VII. The Inhibition, by Malathion, of Thymidine-3H Incorporation by Cells as Affected by NAD and NADH. Addition of cofactors NAD and NADH to malathion (50 ppm) treated cultures showed that at the end of a 4—day incubation the cells in NAD treated cultures showed higher thymidine incorporation than in NADH treated cul- tures. (Figure 22) Experiments were conducted to find out the possible reason for the above observation. Several cell cultures were used and the effect of the cofactors on the hydroly- sis of malathion in cultures were studied. The results indicate that hydrolysis of malathion was greater in NAD treated cultures (Table 6). VIII. Studies to Determine Whether Malathion was Converted to Malaoxon in the Tissue Cultures. Two types of studies were made. In the first study malathion-35$ treated (30-50 ppm) Chinese hamster cell cultures were used. The chloroform fraction was chromato- graphed (electron capture gas-liquid chromatography) and the fractions represented by malathion and malaoxon peaks were collected and assayed for radioactivity. Only 6.8% of the activity, as compared to the malathion fraction, was found in the malaoxon fraction. (Retention times: Malathion - 4 min, 30 sec; malaoxon - 5 min, 50 sec) 80 500T 400‘ I 19.1 I4- 300.. dpm/mg 200 100.- 16 I4- NAD NADH Fig. 22. Effect of cofactors NAD and NADH in malathion treated cultures of Antheria eucalypti cells with regard to thymidine-3H incorporation. No control was set up without cofactors. Incubation period was 4 days. Malathion (99.7%) dosage was 50 ppm. 81 TABLE 6. Radiometric Analysis of the Effect of NAD and NADH on the Hydrolysis of Malathion in Insect Tissue Cultures. Water Fraction Chloroform Fraction (%) (%) M—R egg cells NAD 61.0 31.6 NADH 52.5 47.0 Antheria eucalypti cells NAD 98.0 1.0 NADH 63.1 36.0 Control Modified Grace's 61.0 38.0 Medium (GMM; no (after 4 days) cofactors, and no cells) Note: Malathion-35$ (50 ppm) was used in all the experiments. Incubation period was 8 days. The per- centage activity that is not included in the above figures was contained in the emulsion. 0.2 mM cofactors were placed in the cultures as required. In the second study using an alkaline thermoionic detector (specific to phosphate compounds), the chloroform fraction obtained from the following cultures: Antheria eucalypti, Chinese hamster, human amnion, M-R house fly larval fat body, DDT-R (FC) house fly larval fat body, DDT-R house fly larval fat body, and M-R and DDT—R (FC) house fly embryonic cell cultures, were tested for the presence of malaoxon. No malaoxon peak was detected from 82 any of the cultures. The least amount of malathion de- tectable was 1 ng; the least amount of malaoxon detectable was 34 ng. Maximum amount injected at one time was 20 ng malathion equivalent. However, with the electron capture detector a peak with the retention time identical to malaoxon was consistently observed for chloroform extracts of the malathion treated cultures mentioned above. Per- cent area (sq. mm) of these peaks in relation to the area of the malathion peaks are given in Table 7. The compound represented by the peaks has not been identified. IX. DDT Metabolism in DDT-R and N-R Embryonic Cell Cultures. This study was performed on primary embryonic cell cultures from different strains of house flies. DDT—R strain is highly resistant to DDT and a major metabolite 12.2122 is DDE. The percent conversion of DDT-+DDE in cultures from the two different strains are given in Table 8. TABLE 7. Percent Areas of the Unknown Peaks, as Compared to Malathion Peak Areas, .Cultures. for the Different % Area of Cultures the Unknown Cytotoxic Thymidine-. Peak Effects Incorporation Antheria eucal ti (75 ppm) 0.83 + + M-R embryonic cells (100 ppm) 1.54 - + DDT-R (FC) embry- onic cells Not Not (100 ppm) 10.00 Checked Checked M-R larval fat body cells Not Not (75 ppm) 38.18 Checked Checked DDT-R larval fat body cells Not Not (75 ppm) 38.13 Checked Checked DDT-R (FC) larval fat body cells Not Not (75 ppm) 45.58 Checked Checked Human amnion cells (25 ppm) 47.5 + + Chinese hamster cells (30 ppm) 66.9 + + Notes: Analysis made on GLC (electron capture). Cytotoxic effects: "+" = positive cytotoxic effects 0 cytotoxic effects Thymidine- H incorporation: "+" = inhibition "-" = no inhibition Retention times: Malathion 4 min, Malaoxon 5 min, 5 min, Unknown 30 sec 50 sec 50 sec 84 TABLE 8. Conversion of DDT-+DDE in DDT-resistant and DDT-susceptible Flies. % DDT Remaining in Incubation % DDT Converted Medium + Cells to DDE Control 96.2 3.8 N-R 97.65 0.82 DDT-R 16.22 83.78 Note: SE = 0.75%. Absolute amounts of DDT and DDE represented by the peaks are estimated by using a standard curve based on the amounts injected vs. peak area, 1 ppm DDT was used in the cultures. DISCUSSION 1. "Malathion" Toxicity. The results in general indicate that when malathion was introduced into the cell cultures, it brought about reduction in DNA, protein, and RNA syntheses, and the number of cells that entered anaphase (population in- crease). Quantitative variation in relation to the ori- gin of the cells and the dosage of malathion administered was observed. Qualitative differences based on cyto- pathological effects were also evident. The common para— meter used for comparison is one generation time which is presumed to give metabolic equality in terms of cell du- plication. One generation time for Antheria eucalypti cells was approximately 4 days (Figures 1 and 2), and one generation time for mammalian cells was 20—30 hours. Mitsuhashi gt gt. (1970a) demonstrated that mala- thion inhibited the growth of Antheria eucalypti cells at approximately 10 ppm dosage. Cytopathogenic symptoms and reduction in growth were observed after 4 days. At 100 ppm, although some growth suppression was noticed even before the 4th day, significant mortality occurred 85 86 only after the 4th day. At 1000 ppm dosage range their cultures showed extensive cell mortality. It is their contention that malathion probably accumulated in the cells over the period of 4 days and presumably reached a critical concentration by the end of the 4 day period and caused spontaneous cell mortality. The response of Antheria eucalypti cells to malathion in our experiments at 50-150 ppm dosage range, in terms of cytopathogenicity was similar to their observations (Table 3; also vide page 53). Significant cytopathogenic symptoms were gen— erally observed only after 4 days, although at 150 ppm the cells showed certain degree of shriveling and granu- lation even by the end of the third day. However, our results seem to show that at 75 ppm, some of the basic biochemical processes related to growth and cell division were affected significantly even before any cytopathologi- cal effects were noticed. A 45% reduction of DNA syn- thesis was noticed by the end of 72 hours (Table 1). In Chinese hamster cell cultures detachment of cells from the surface of the flask occurred 12-18 hours after the treatment of 30-80 ppm (80 ppm was the maximum dosage tested; vide page 58)- Vacuolations also appeared around that time. However, the effect of "malathion" was evi- dent even at 25 ppm dosage by the end of 5 hours as de- termined by the number of cells that entered anaphase. A reduction of 50% in the number of mitotic cells was 87 noticed (Figure 13) even before any cytopathological symptoms could be observed. These observations seem to suggest that malathion or its toxic metabolite(s) probably affected the cellular metabolism progressively starting soon after the treatment. The cytopathological effects appeared after the inhibition of certain cellular meta- bolic process(es), such as protein, DNA, and RNA syn- theses, had reached a certain critical level(s). Occur- rences of such irreversible physiological damages pre- ceding "cell death" and cytopathological effects have been reported by Ward and Plageman (1969). A comparison of the "malathion" tolerance levels, with regard to DNA synthesis, cytopathological effects, cell mortality, and growth inhibition in terms of protein content measurements between our insect cell cultures and mammalian cell cultures shows a striking difference be- tween the two groups (Figures 10, 11; Tables 2, 3). Ap- proximately 50% inhibition was caused by 21 ppm and 41 ppm malathion in human amnion cells and Chinese hamster cells, respectively, and 89 ppm and 113 ppm in Antheria eucalypti cell cultures and M-R house fly embryonic cell cultures, respectively. These comparisons are made on the basis of inhibition brought about approximately in one generation time (doubling time) of the cells which is 20-30 hours for mammalian cells and 96-108 hours for Antheria eucalypti cells. No doubling time was determined 88 for house fly embryonic cells. Thus with regard to DNA synthesis at 50% inhibition level, the malathion dosage difference was 100%. With regard to total cell de- struction, the difference was about 200%. It is also true that there are differences in tolerance levels to malathion even between the different mammalian cell cultures. Based on cytopathological effects and growth inhibition the toxic dose 50% (TDSO) and inhibitory dose 50% (IDSO) for HeLa cells and liver cells were shown to be 20 and 13 ppm and 10 and 15 ppm, respectively (Gabliks and Friedman, 1965). In our human amnion cell cultures an inhibition of 46% was imposed by 25 ppm malathion. However, the focus here is on the wide difference in the tolerance levels between the mammalian cell culture group and the insect cell culture group, which is more than 100% with regard to "malathion" inhibition of DNA synthesis and "malathion induced" cell mortality. The wide difference in tolerance between the insect cells and the mammalian cells is significant for the reason that in terms of whole animals, insects have been shown to be far less tolerant to malathion than mammals, which is the reverse of what we found in our experiments. The dose 50% (LDSO) for mice was 815 ug/gram (oral) and 12 pg/gram (topical) for house flies (O'Brien, 1960). One of the reasons for the reversal of malathion tolerance between cell cultures and whole animals may be sought in the mode of action of the 89 compound in the two different systems. Most of the acute toxic effects in whole animals were shown to be due to the inhibition of acetylcholinesterase in the nervous system by malaoxon which was produced by enzymatic de- sulfuration of malathion (oxidation; O'Brien, 1960, 1967). The high carboxyesterase activity in mammals which was responsible for degrading malaoxon was shown to be par- tially responsible for the high tolerance levels observed in mammals. However, suggestions as to the possibility of another enzyme or enzymes undergoing parallel inhibi- tion has been offered by O'Brien (1960). Greenberg and LaHam (1969, 1970) showed that there was no correlation between acetylcholinesterase inhibition by malathion and malathion induced teratogenic effects in chick embryos. Malathion was shown to inhibit the uptake of tryptophane from the yolk by the embryo, and concomitant reduction in protein synthesis, general growth reduction, and mal- formation. When certain compounds with g values similar to tryptophane was injected into the embryo, normal em- bryos were produced thus indicating that reduction in tryptophane level in the embryo caused some kind of elec- tronic imbalance which led to the induction of teratogenic effects. Tryptophane has a 5 value indicative of higher electron donating ability. When NAD, to which tryptophane is a precursor, was injected into the embryo, there was a significant reduction of malformation but still there was 90 a certain amount of growth retardation. Further in our experiments up to 20 ppm (maximum dosage tested) malaoxon did not cause any significant ill effects with regard to any of the metabolic processes examined (Figures 19, 20, and 21). No malaoxon was detected in the chloroform ex- tracts of any of the cultures which were affected signi- ficantly by malathion. If malaoxon was responsible for the inhibitory effects noticed it should do so at con- centration above 20 ppm. However, when the chloroform extract of radioactive malathion (30 ppm) treated Chinese hamster cell culture tested for malaoxon by collecting at the malaoxon retention time and counting the radio- activity, only about 7% activity was found. In our final solution prepared for gas-liquid chromatography analysis, if the cultures had converted 20 ppm malathion to mala- oxon, there should be at least 24 ng/ul malaoxon after allowing 70% immediate hydrolysis. However, no malaoxon peak was observed even with 4 pl injection (96 ngmala- oxon equivalent). The thermoionic detector, sensitive to malaoxon at approximately 36 ng, was used. If malaoxon conversion did take place in our cultures, it probably was hydrolyzed immediately and almost completely. Such rapid hydrolysis is a resistance mechanism in mammals (O'Brien, 1960). Based on such observations it appears that no significant malaoxon conversion did take place in 91 our cultures, and if it did, it did not build up to a significant level to cause any ill effects on cells. Extreme purification of malathion samples used did not reduce the toxicity of malathion (Figure 21). Based on the above information it appears that the inhibitory effects and cytopathological effects observed were caused by malathion and not through conversion to malaoxon. However, the possibility of an impurity such as Mx4 (Table 5; Figure 21) which would be chromatographed with our malathion sample and contributed to the malathion toxicity cannot be eliminated. On the other hand, since there was no difference between the purified malathion and unpurified malathion (99.7%) as to their toxicity, the toxicity con- tributed by the unknown compound may not be significant. Wilson and Walker (1966) showed mercaptosuccinate to be as toxic as malathion. Mercaptossucinate SH-CH-COOH CHZ-COOH We do not know at this point if any mercaptosuccinate was produced in the system and the toxicity was due to such a metabolite. Table 2 indicates a general relationship between the levels of toxicity of malathion to the insect cells and to the mammalian cells. The unknown peak area is higher for mammalian cell cultures which showed less resistance 92 and low for insect cell cultures which showed higher re- sistance to malathion. Even within the insect cell cul- ture group the peak value is higher for embryonic cells of DDT-R strain than the embryonic cells of M-R strain. The significance of the peak is not known. It may repre- sent a breakdown product of the cell due to cell de— generation caused by malathion or it may be a toxic meta- bolite of malathion. It is a chloroform extractable product and on that basis is not mercaptosuccinate. Rea- sons have already been given to show (vide page 78) that it cannot be malaoxon. Table 6 indicates when NAD was added to cell cultures, malathion was converted to water soluble compounds at a much faster rate. Higher DNA syn— thesis was noticed in such cultures (Figure 22). This supports our initial contention that the toxic effects observed were due to malathion itself. II. Interrelationship Between DNA, RNA, Protein and Cell Division Inhibitors. From our results it is not possible to tell the exact site of biochemical 1esion(s) caused by malathion which resulted in the various inhibitory and cytOpatho- logical effects noticed. Neither is it possible to enu- merate the sequence in which these events occurred in our cultures. An attempt to unravel the relationship between these effects may be attempted in the light of previous works by other researchers. 93 A strong dependence of DNA synthesis on protein syn- thesis was demonstrated by several workers in tissue cul- tures (Harris, 1959b; Watts and Harris, 1959; Weiss, 1968). Quantitative relationship between DNA synthesis and pro- tein synthesis was shown by Seed (1965) in several mam- malian cell cultures. In Euplotes extensive incorporation of histidine was shown to occur at the place of DNA syn- thesis (Prescott and Kimball, 1961). However, protein synthesis was not dependent on DNA synthesis. When DNA synthesis was inhibited, protein synthesis was not af- fected (Sheek gt gt., 1960; Levintow and Eagle, 1961). Based on these findings it seems likely that protein probably was the first general cell component to be af- fected which in turn affected the DNA synthesis (Figure 10). It is possible that malathion was relatively more specific to synthesis of proteins which are closely re— lated to DNA synthesis such as histones (Lehninger, 1970). Prescott and Kimball (1961) demonstrated the presence of proteins rich in histidine to be closely associated with DNA synthesis. The possibility of relatively higher in- hibition of enzymes related to DNA synthesis cannot be excluded. Malathion and malaoxon inhibition of esterases other than acetylcholinesterase had been demonstrated by Ecobichon and Kalow (1963). Between DNA synthesis and RNA synthesis, and RNA synthesis and protein synthesis in mammalian cell cultures, no mandatory coupling was 94 observed (Harris, 1960). RNA synthesis was shown to con- tinue even in the absence of DNA synthesis (Watts and Harris, 1959; Feinendegen gt_gt., 1960). In the macro- molecules of Euplotes DNA synthesis was shown to proceed in the absence of RNA or RNA synthesis (Prescott and Kimball, 1961). RNA synthesis was inhibited without in- hibiting protein synthesis (Harris, 1960). Seed (1965) and Studzinski and Lambert (1968), however, showed con- current RNA and protein synthesis. This observation does not necessarily indicate a coupling of RNA and protein synthesis. Thus the relationship between the inhibition of RNA synthesis and the rest is not very clear. RNA probably was inhibited independently or the inhibition was a result of overall degeneration of the cell that had set in as a result of previously discussed inhibitory effects. The relatively low level of inhibition of RNA synthesis (Figure 10) may be a result of relatively late onset of RNA synthesis inhibition. The following possi- bilities, however, cannot be overlooked: l. Malathion did not have as much effect on RNA as it had on DNA and proteins because these systems had a "differential susceptibility" to differ- ent toxic agents, as reflected in the works of Harris (1960) and Prescott and Kimball (1961). 2. The inhibition was not imposed on all species of RNA as shown for the alkaloid poisons (Wagner 95 and Roizman, 1968), and as a result inhibition of relatively a small portion of the RNA did not significantly reflect in the overall observa— tions. III. Mitotic Arrest and Cell Death. It is evident from Figure 13 that the Chinese ham- ster cells suffered a significant mitotic arrest before the M phase in malathion treated cultures under the given experimental conditions. A comparison of Figure 13 and Figure 11 indicate a higher sensitivity of the cells to malathion with regard to mitotic arrest than with regard to DNA synthesis. A similar sensitivity difference was shown by others (Levintow and Eagle, 1961). A certain amount of irradiation led to total mitotic block but allowed DNA, RNA, and protein syntheses to proceed at re- duced levels. ~At 25 ppm dosage the mitotic arrest was ”50% in 5 hours and the DNA synthesis inhibition was less than 20% in 24-30 hours. At 40 ppm the mitotic arrest was more than 80% within 10 hours while DNA synthesis in- hibition was 345% after 24 hours. Based on the above similarity one cannot positively identify malathion as a "radioimetic" compound. Loveless and Revell (1949) in- cluded chromosomal aberrations such as breaks and the alkylating property as two of the important characteris- tics of radioimetic compounds. Our studies did not 96 include the above two aspects. However, certain organo- phosphates are said to cause nucleoprotein alkylation. More information is needed to ascertain the mutagenic significance of malathion. Hansen and Vandevoorde (1966) observed that in their mammalian cell cultures azaserine at certain dosages com- pletely stopped the cells from dividing, but without in- hibiting the DNA synthesis completely. There was a lag in DNA synthesis but the synthesis proceeded at a low level and doubled and tripled with the cells undergoing no division. Based on the difference between the sensi— tivity of the cells in our cultures in terms of DNA syn- thesis inhibition and mitotic arrest, it seems possible that inhibition of protein synthesis resulted in the in— hibition of enzymes or other proteins involved in the mitotic process leading to the arrest of mitosis prior to anaphase. The rapid cell death, according to Hansen and Vandevoorde, was due to the blockage of DNA synthesis at dosages above the cell division inhibitory dosage. In general this observation, as has already been shown, is similar to what we observed in our experiments (Figures 11, 13). The cells which showed cytopathological conditions, when stained with Feulgen stain, showed nuclear dis- integration to have taken place (Figures 5, 6). On the other hand nuclear disintegration also occurs in cells 97 degenerating because of "aging." On the above terms, "aging" was relatively high in malathion intoxicated cul- tures. According to Hansen and Vandevoorde the cell death was due to increase in size of the nuclei, due to continued synthesis of DNA with concurrent mitotic ar— rest, which ultimately led to the breaking up of the nuclei and cell death. The final cell death could have happened in our cultures the same way. The enlargement of the nuclei in malathion treated Chinese hamster cells is evident from Figures 15 and 16. IV. The Effect of NAD and NADH on "Malathion Effect." A study on the genetic control of insecticide chemi- cal metabolism in house flies showed that NADPH-dependent oxidases played a significant part in the oxidative metabolism of various insecticide chemicals. With the exception of malathion, the resistance was shown to be directly proportional to the activity of the oxidases (Plapp and Casida, 1969). (Oxidases mainly potentiate malathion by converting it to malaoxon which is the most potent acetylcholinesterase inhibitor of the two (O'Brien, 1960, 1967; Plapp and Casida, 1969). Enzymic degradation in mammals is effected especially by carboxyesterase enzyme(s) (O'Brien, 1960; Matsumura and Brown, 1961). In general the carboxyesterase action brings about the follow— ing degradation: (O'Brien, 1967) 98 CH 0 P S SCHCOOC H CH 0 P S S HCOOC H (3)2() 25 mammals;(3)2()i 25 CHZCOOCZH5 and /' CH COOH ’ insects malathion acid urinary product in J mammals ./ \i/ 1nsects (CH30)2P(S)SiHCOOH CH 0 P S OH H ( 3 )2 ( ) C 2COOH dimethyl phosphorothioate malathion diacid Matsumura and Brown (1961) demonstrated that in EElEE tarsalis the resistance is also primarily due to the high- er carboxyesterase activity which hydrolized malathion. Their work indicated NAD to be the required cofactor for carboxyesterase. Based on these observations it appears that in our experiments, the difference in the level of malathion observed at the end of the incubation period of NAD and NADH treated Antheria eucalypti cell cultures (Table 6) may be due to enhanced carboxyesterase activity. Further, it seems that in Antheria eucalypti cell cul- tures, the hydrolysis of malathion was dependent on the ability of the cells to synthesize the cofactor, rather than the ability to synthesize the enzyme, as was shown with regard to Culex tarsalis by Matsumura and Brown (1961). This contention can be supported by the work of Greenberg and LaHam (1969, 1970), in which malathion in- duced teratism in chicks was partially rectified by in- jecting NAD. These observations implied that a mutation 99 leading to increased NAD production will increase the re- sistance. The reduction in malathion level in our cul- tures showed correlated resistance to malathion toxicity as measured in terms of DNA synthesis (Table 6; Figure 22). In malathion resistant embryonic cell cultures, the difference between NAD and NADH treated cultures was not as pronounced as was found in Antheria eucalypti cell cul- tures. There was also no inhibition of DNA synthesis. Since embryonic cells from resistant strains did not de- grade malathion to malaoxon in greater quantities than susceptible strains, it is possible that the carboxy- esterase system had not developed extensively at this time. The resistance found in our cultures seem to be dependent on a different gene or genes from the one shown for Culex tarsalis by Matsumura and Brown (1961). Thus the mechanism of resistance to organophosphorus insecti- cides seem to be highly complex. V. Musca domestica Fat Body Cell Cultures. An analogy has repeatedly been drawn between insect fat bodies and the mammalian liver. The reason for such a comparison is evident when various fat body related metabolic processes are taken into consideration (Kilby, 1963). Fat bodies are shown to be concerned with the various intermediary metabolism of carbohydrates, pro- teins, lipids, amino acids, purines, pteridines, and 100 various other insect pigments. Its significance in the detoxification process is emphasized. It is also a storage organ for protein, glycogen, and fats (Kilby, 1963 Wigglesworth, 1965). The possible use of fat body tis- sues tt_ytttg for studying the effect of malathion was investigated. DNA synthesis as studied in terms of thymidine-3H incorporation (vide page 59; Figure 24, Appendix) and cell autoradiography was shown to be not significant in our fat body cell cultures tg_ttttg. Ac- cording to Kilby (1963), breakdown products of fat body cells did not give any Feulgen positive reaction for nucleoproteins. This is probably because the fat body cells are not highly dividing cells. However, Wiggles- worth (1965) indicated that they undergo division during larval moulting. In our cultures no cell division was noticed. This prevented us from using fat body cells for studies related to DNA synthesis. Our histological tests on 4-day-old cultures gave positive response for mito- chondrial enzymes such as glutamic dehydrogenase, iso— citric dehydrogenase, and a-glycerophosphate dehy— drogenase which is responsible for reoxidation of NADH in the EMbden-Meyerhof sequences in insects. £§_ytzg metabolic activity of fat bodies increases with insect metamorphosis and moulting. A cyclic pat- tern in relation to the ovarian cycle with regard to RNA content of the fat bodies was observed. Blood protein 101 content varied accordingly. This activity was shown to be related to the hormone secretory activity of the neuro— secretory cells (Hill, 1962; Highman gt gt., 1963; Chen, 1959; Salma, 1964; Mills, 1966; Mills gt gt., 1966). Based on this information it seems possible that insect fat body cell cultures may be a good biochemical tool for the study of detoxification mechanism (Perry, 1964), intermediary metabolism, and the related hormonal interplay. VI. The Metabolism of DDT in DDT-R and N-R Embryonic Cell Cultures. This particular study, with regard to DDT conver- sion to DDE in house fly embryonic cell cultures of the DDT-R and N-R strains, was done primarily in order to investigate the possibility of using such tg_!tttg cul- ture systems in the study of various genetic aspects of the animals with regard to pesticide resistance and metabolism. Attempts have been made by several workers to study £2.21E52 the genetics involved in pesticide resistance by using tissue culture as a tool. Tamura (1958) showed the existence of differences with regard to parathion re- sistance in the different strains of Drosophila melano- gaster. Single gene mutations in cell cultures were studied by Kao and Puck (1969) with regard to nutritional 102 requirements. Mutagenic properties of chemicals were studied on eye-antennal disk cultures with regard to the formation of pigments (Schneider, 1964). However, the results obtained by us with regard to the effect of malathion on the primary embryonic cell cultures of the malathion resistant and non-resistant house fly strains did not bring out any significant difference between the two cultures. The results were not adequate to ascertain whether the genetic differences exhibited by the adult flies with regard_to malathion resistance was reflected in some way in the embryonic cells (vide page 55-59). No detectable conversion of malathion to malaoxon was noticed with either of the cell cultures. The significance of the difference between the M-R embryonic cell cultures and DDT-R (FC) embryonic cell cultures with regard to the area of the "unknown peak" (Table 7) has not been yet ascer- tained. Hence, we proceeded to use embryonic cells of DDT-R and N-R strains to find out if those cells might reveal any genetic differences with regard to DDT metab- olism as shown by the adult flies. Metabolism of DDT has been shown to follow differ- ent courses in different animals. In mammals DDT metab- olism was reflected in species variation. In insects the best known metabolite of DDT is DDE (O'Brien, 1967). The enzyme responsible for this reaction was shown to be DDT-dehydrochlorinase. In our cultures a significant 103 difference in the enzyme activity between the embryonic cells of the DDT-R strain and the N-R strain was noticed (Table 8) indicating the resistance mechanism to be op- erative even at this very early stage of development. The difference in percent conversion of DDT to DDE between our embryonic cell cultures of N-R strain and DDT-R strain was about 82%. In adults it was shown to be about 50% (Perry, 1964). However, the mortality difference was 98% in 24 hours in the case of the latter. As it is evident from the data of.Perry (1964), the percent con- version of DDT-»DDE seems to be related to enzyme titre in the flies. While percent mortality was the same in the different resistant strains, the percent conversion differed up to 20%. The reason for the high conversion of DDT-+DDE in our cultures may be due to various fac- tors. DDT in general is said to enter into lipoprotein structure of the nerve membrane, cause ionic imbalance leading to tremors, paralysis and death. It has also been shown that DDT causes partial inhibition of cyto-2 chrome exidase and it is indicated that the adults of some DDT-resistant strains of the house fly contained higher titers of enzyme or a more active flavoprotein electron transport system that the susceptible flies. It was suggested that DDT-resistance may be partially re- lated to the fly's ability to maintain a higher energy yielding process (Sacktor, 1950, 1951). Based on 104 correlations between DDT-resistance and cytochrome oxidase activity, Perry (1964) concludes that at the present time there is not enough evidence to show that the toxic effect of DDT in house flies is due to the inhibition of any vital enzymes. Wang gt gt. (1970) demonstrated that in culture tissue cells of Aedes aegypti, DDT at 5 ppm dosage level brought about a decline in protein content of the cells and stimulated protein synthesis at a dosage of 1 ppm and less. It was indicated that the increase in protein synthesis was a result of stimulation of micro- somal hydroxylation enzyme. Our DDT dosage level was 1 ppm. Further, no ap- parent development of the nervous system was noticed in our cultures. The contracting cells were identified as muscle cells (Paul, 1965). No inhibition of contractions was noticed either with malathion or with DDT. Based on these observations it seems possible there might have been a slight stimulation of enzyme activity in the embryonic cell cultures of DDT-R. Such increases in enzyme activity at low levels of a toxic material has been demonstrated in the case of plants with regard to the effect of sima— zine. The effect of DDT on the embryonic cells of the non-resistant flies is not known. It is apparent, since no glutathion was provided in the medium, that the embryonic cells did contain the glu- tathion synthesizing mechanism, at least in the cells of 105 DDT-R strain. The difference in conversion of DDT +DDE may partially be related to the titre of the DDT- dehydrochlorinase enzyme and/or the amount of cofactor synthesized. While enzymes responsible for DDT-resistance had been initiated at this early embryonic level, enzymes responsible for malathion resistance in adults were not noticed. Carboxyesterases seem to come into play much later in the life cycle than DDT-dehydrochlorinases. It should be mentioned at this point that our study, with regard to DDT conversion to DDE in house fly embryonic cell cultures of the two different strains, was done primarily in order to investigate the possibility of using such tg_!tttg culture systems in the study of various genetic aspects of the animals. From our work it becomes clear that primary embryonic cell cultures of house flies did reflect the genetic make-up of the strain with regard to DDT—resistance. It seems that this cul- ture system can be effectively used for effects of DDT other than its effects on nerves. The advantage of this experimental system is that a minimal amount of barriers are present between the chemi- cal and the cell. The extent of cell/chemical contact can be assumed to be comparable in all the samples. The in- fluence of various other organs of the body as confronted in the case of studies 12 vivo in adult is eliminated. SUMMARY The interaction of malathion and various tissue culture cells of insect and mammalian origin was studied. The introduction of malathion into the cell cultures brought about an inhibition of DNA, protein, and RNA syn- theses, and the number of cells that entered anaphase (pOpulation increase). CytOpathological effects appeared after the inhibitory effects had reached a certain level(s). A comparison of the "malathion" tolerance levels with regard to DNA synthesis, cytopathological effects, cell mortality, and growth inhibition showed a wide dif— ference between the insect cell culture group and the mammalian cell culture group. The insect cells showed a 100% higher tolerance in terms of "malathion inhibition" of DNA synthesis and "malathion induced" cell mortality. This difference is significant for the reason that, in terms of whole animals, insects have been shown to be far less tolerant to malathion than mammals, a reason for the popularity of the pesticide. 106 107 Malaoxon, the toxic metabolite of malathion 12.2122! did not cause any observable effects on cells. Concen- trations up to 20 ppm were studied. No malaoxon was de— tected in our malathion intoxicated cultures. Probably there was no conversion of malathion to malaoxon by the cells. Comparison of the effect of 99.7% pure analytical grade malathion and further purified (100%) malathion did not significantly vary as to their toxic effects on cells. However, the possibility of contribution of toxicity by undetected impurities and certain metabolites, though deemed insignificant, were not eliminated. With regard to the sequence of malathion induced inhibitions, proteins are suspected to be the first gen- eral cell component affected. Possible inhibition of enzyme(s) or proteins related to DNA synthesis is sus- pected to have brought about the inhibition of DNA syn- thesis. RNA synthesis was either inhibited independently or resulted as a consequence of the overall degeneration of the cell. The relatively low level of inhibition may be due to a certain amount of "RNA species specificity" of malathion or the late onset of inhibition. The Chinese hamster cells showed a higher sensi- tivity to mitotic arrest than to inhibition of DNA 108 synthesis. The cells seem to have been arrested before the M phase. The probably interactions in the malathion induced inhibitions seem to be as follows: Protein inhibition-—+DNA synthesis inhibition .t. "Malathion"4’///a : ;: Mitotic inhibition + RNA synthesis 1, n . " inhibition ' Aging Cell death Arrest of cell duplication with continued low level of DNA synthesis could have brought about the nuclear dis- integration and final cell death. In general the dif- ferent inhibitions led to the premature "aging" of the cells. NAD enhanced the hydrolysis of malathion in Antheria eucalypti cell cultures. Enhanced carboxyesterase activ- ity due to the addition of NAD is suspected. It is specu- lated that a mutation which increases the synthesis of NAD would increase the resistance in the cell cultures. DNA synthesis and cell division was not observed in fat body cells of Musca domestica t2 vitro. But they showed significant protein synthesis and the presence of various enzymes were evident after the test period of 4 days, when kept in Grace's modified medium. 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Musca domestica fat body cells. Glutamic dehydrogenase. Magnification 396x. No counter stain. 128 Fig. 25. Musca domestica fat body cells. a- glycerophosphate dehydrogenase. Magnification 792x. No counter stain. 129 Fig. 26. Musca domestica fat body cells. Isocitric dehydrogenase. Magnification 396x. No counter stain. JIH-I "11111111111111.1118“