THE CYTOLOGICAL EFFECTS OF PESTICIDES 0N HUM-AN . CHROMOSOMES Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY DAVID HENRY KRAUS-E 1970 ABSTRACT THE CYTOLOGICAL EFFECTS OF PESTICIDES ON HUMAN CHROMOSOMES By DAVID HENRY KRAUSE Chromosomes of human peripheral leucocyte cells were analyzed for cytological damage attributable to pesticide usage. Widespread introduction of pesticides into the environment makes contact unavoidable and necessitates the development of an in 1119 human monitoring system. Circulating leucocytes offer a convenient and sensitive indicator of potential genetic effects in humans. Cells, exposed in 1139, were obtained from high- use Sprayers of pesticides and cultured in vitgg, Slides were prepared from these cultures and mic- roscopically analyzed for chromosome aberrations. Selected pesticides were screened, using human amnion AV3 cells, for effects on chromosome breakage and cell growth. The incidence of chromosome breakage in the ex- posed group was slightly higher than in the control group. However, the data was inconclusive and further study was indicated. In the AV3 tissue culture ex- periment, captan was shown to be of concern due to its increase of chromosome breakage and inhibition of cell growth. THE CYTOLOGICAL EFFECTS OF PESTICIDES ON HUMAN CHROMOSOMES BY DAVID HENRY KRAUSE A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1970 C5» ”- (/75 / "If “:7 #1:"?! AC KNOW LE DGMFNTS The author would like to express his gratitude and appre- ciation to Dr. R.A. Hoopingarner, Dr. N.C. Leeling, and Dr. J. E. Trosko for their assistance and counsel throughout the course of this investigation. Also, I wish to thank Arthur W. Bloomer and Adrian Oudbier for their help in obtaining the financial and technical assistance which made the project possible. The project was funded by the Michigan Department of Public Health, through the United States Department of Public Health. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O Page 0 O O O O O O O O O O O O O O O 1 MATERIALS AND METHODS .’ . . . . . . . . . . . . . RESULTS 0 O O O O O 0 DISCUSSION. . . . . . . . . SUMMARY......... RECOMMENDATIONS . . . . BIBLIOGRAPHY . . . . . . . iii .17 ..24 36 39 4O 41 LIST OF TABLES Table - Page 1 Effect of pesticides on chromosome aberration . . . 26 2 Effect of pesticides on chromosome number . . . . 27 3 Distribution of exposed group to pesticides encountered.....................28 4 Acute oral toxicities of pesticides encountered by ' exposed group O O O O O O O O O O O O O O O O O O O O 30 5 Effect of pesticides on chromosomes of human peripheral leucocyte cells i_n_vitro . . . . . . . . . 31 6 Effect of pesticides on chromosomes of human peripheral leucocyte cells _i_r_1_vitro . . . . . . . . . 32 7 Effect of pesticides on chromosomes of AV3 cells . 33 8 Effect of pesticides on growth of AV3 cells . . . . .34 iv LIST OF FIGURES Figure Page 1 Normal human chromosome metaphase plate . . . 21. 2 Break with associated fragment in a human 22 chromosome.................... 3 Cap inahuman chromosome . . . . . . . . . . . 23 4 Comparative exposure of user group to frequently encountered pesticides . . . . . . . . . . . . . . . 29 5 Effect of pesticides on growth of AV3 cells . . . . 35 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. NOME NC LATU RE Acaralate Alar Arnitrole BHC Captan Carbaryl Colcemide Cyprex DDT Fe rbam Guthion HEOD Irnidan Karathane Lead Arsenate Lime Sulfur Maneb Napthaleneacetic acid Oil Paraquat Parathion Phaltan Phosphamid on Phygon Sima zine Sulfur Streptomycin vi Is Opropyl 4, 4' -diclorobenzilate Succi'nic acid 2, 2-dimethylhydrazide 3 -Amino-l, 2, 4-triazole 1, 2, 3, 4, 5, 6-Hexaclorocyclohexane N- Trichloromethylthio -4 - cyclohexene - 1, 2 -dicarboximide l - Naphthyl N-methylcarb arnate N-Desacetyl-N-Methylcolchicine Dodecylguanidine acetate 1,1,l-Trichloro-2, 2 -bis(p-chlor0phenyl) ethane — Ferric dimethyl dithiocarbamate O, 0-dimethyl-s -ph08phorodithioate l, 2, 3, 4, 10, 10-Hexachloro-6, 7-epoxy- 1, 4, 4a, 5, 6, 7, 8, 8a-octahydro-1, 4-endo- exo - 5 , 8 -dimethanonaphthalene N-(Mercaptomethyl)phtha1imide S -(0, 0 - dimethyl phosphorodithioate) 2 - (l-Methylheptyl) -4, 6 -dinitrophenyl crotonate Dibasic lead arsenate Calcium polysulfides and calcium thiosulfate Manganous ethylene bisdithiocarbamate l—Napthaleneacetic acid 1, 1' -Dimethyl-4, 4' -bipyridinium dichloride 0 , O - Diethyl 0 -p -nitr0pheny1 phosphorothiofie N- (T richloromethylthio) -phthalimide 2 - Chloro -2 -diethylcarbamoy1 -1- methylvinyl dimethyl phosphate 2 , 3 - Dichloro-l, 4 -naphthoquinone 2 -chloro-4, 6 -bis(ethy1amino) -s -triazine Sulfur 2 , 4- Diguanidino-3 , 5, 6 -trihydroxycyclo - henyl 5-deoxy-2 -O-(2 -deoxy-2 -methyl- amino-g - glucopyranosyl)—3-formy1 pentofuranoside 28. 29. 30. 31. Systox TEPP Thiodan Zineb vii 2:1 0, O-diethyl-O-AND S-2—(ethylthio) ethyl phosphorothioate (thiono isomer) and 0, 0-diethyl-S-2-(ethy1thio)ethy1 phos oho rothioate ( thiol is ome r) Tetraethyl pyrophosphate 6, 7, 8, 9, 10,10-Hexachloro—l, 5, 5a., 6, 9, 9a- hexahydro-6, 9-methano-2, 4, 3-benzo- dioxathiepin- 3 - oxid e Zinc ethylene bisdithiocarbamate INTRODUCTION. During the past decade there has been increasing concern over the effects of the introduction of pesticides into our environment. Some pesticides are mutagenic and the normal tests for mammalian tolerance may be an insufficient clearance. A risk to mankind much greater than acute, subacute, or chronic toxicity may exist in subtleflgenetic effects of these chemicals. Pesticides as chemical mutagens are a hazard which has not been systematically investigated. The protection of public health depends upon determining the nature and severity of harm that may be caused by these agents. Chromosomes from human peripheral blood offer a convenient and sensitive_ili ELY? monitoring system for estimating potential genetic damage in man. The study of human chromosomes dates back almost a century, however it has only been in the last ten years that culture techniques have been made available to make analysis of large numbers of cells feasible. In fact, it was not until 1956 that the correct diploid number of somatic chromosomes in man was established. The first attempts to visualize and count the chromosomes of man were in 1882 by Flemming and in 1891 by Hansemann (Robinson, 1961). Due to poor techniques estimates varied greatly until 1923 when it was concluded by Painter, and generally agreed upon for the next 33 years by the majority of in- 2 vestigators, that the diploid number of chromosomes in a human somatic cell was 48. During this period an important discovery led to determin- ation of the correct human diploid chromosomal number. In 1952 Hsu and Pomerat and, simultaneously, Huges reported that a cell in metaphase swells when placed in a hypotonic citrate solution before fixing and staining. This excessive intake of water by the cells allows the chromosomes to separate making analysis of them easier. Using this new technique Tjio and Levan (1956) established a diploid somatic chromosome number of 46 for man, 44 autosomes and 2 sex chromosomes. This finding was confirmed by Ford and Hamerton (1956), Tjio and Puck (1958) and Chu and Giles (1959). Three other techniques aided the rapid advance in human cyto- genetics. Human chromosomes are analyzed at the metaphase stage of mitosis, however it is only by chance that one finds a cell in this stage. Ford gal. (1956) and Tjio and Levan (1956) made use of the action of colchicine as a mitotic blocking agent to hold cells at the metaphase stage of division by inhibiting Spindle fiber formation. The addition of this chemical to culture media not only increased the number of cells suitable for analysis, but also somewhat shortened and dispersed the individual chromosomes. Still, chromosome work on humans depended upon bone-marrow puncture or skin b10psy for material and upon classic squashing methods. These problems were alleviated, when in 1958 Rothfels and Siminovitch develOped an air-drying method. The advantages of air drying of meta- phase cells cultured on glass are that it provides well-spread chromo- somos in one focal plane and there is a minimum of over-lay and . scattering. In 1960 Nowell made an important discovery which per- mitted convenient chromosome analysis of a significant number of cells. He found that phytohemagglutinin would induce normal peripheral leucucytes from human blood to enter DNA systhesis and division. Since under normal conditions in the body only a very small percentage of the cells are undergoing mitosis, this finding was of great significance to chromosome analysis. The use of peripheral blood offered a material that was easily obtained with a minimum of discomfort. A classic paper written by Moorhead gt_a_1.in 1960 combined the afore- mentioned techniques to provide a convenient method of culturing human leucocytes for critical analysis of chromosome morphology. Since the development of these tissue culture techniques and Moorhead's work, many slight modifications and simplifications have been made for the culturing of white cells from peripheral blood (Steinberger, 1964, Bishun, 1965, Uchida, 1966, Katz, 1970). Even though the study of human chromosomes dates back to 1882, it has only been in the last decade that practical and convenient methods have been developed to allow a detailed visual study of the number and morphology of human chromosomes. Since 1959 when Lejeune (Hampton, 1964) demonstrated a chromosomal abnormality resulting in mongolism, there has been no doubt as to the clinical value of chromosome examination. In order to effectively use these methods in 4 the interpretation of chromosome aberration data, an understanding of chromosome morphology and cell division is necessary. Using tissue culture techniques, the infrequently dividing cells of most normal body tissues undergo division in significant numbers. The cell cycle encompasses the time from the beginning of the growth phase to the end of mitosis. The period of non—division i. e. ”no growth" is termed G0 referring to the time'after mitosis occurs but before the onset of growth processes. G1 is a presynthetic period; a period in which net cell growth (protein, RNA, etc. ) preceeds, as well as preparation for DNA synthesis. The second stage of the cell cycle is S or the DNA synthetic period. Last is the post-DNA synthetic period, G2, followed by mitosis, M (Gilbert, 1964). Mitosis is divided further into four stages: prophase, when the chromatin begins to condense and spindle fibers begin to form; metaphase, when the nuclear membrane dis- appears and the chromosomes shorten and line up on the equatorial plate; anaphase, when the doubled chromosomes Split and half of each migrates to each pole; and telophase, when nuclear membrane reforms, chromo- somes elongate and the two daughter cells are formed. The cell then goes into interphase which includes GO, 01' S, and G2. Chromosomes are the carriers of the hereditary elements of cells. A normal human cell contains 46 chromosomes. They are paired, 22 pairs of autosomes and 1 pair of sex chromosomes. These chromosomes were classified into easily identifiable groups in 1960 at the University of Colorado in Denver (Book flai, 1960). By this classification there 5 are five groups: Group 1-3, large chromosomes with approximately median centromeres; Group 4—5, large chromosomes with sub- median centromeres; Group 6-12, medium sized chromosomes with submedian centromeres which resemble the X chromosome; Group 13-15, medium sized chromosomes with nearly terminal centromeres; Group 16-18, short chromosomes with submedian centromeres (approx- imately median in 16); Group 19-20, short with approximately median centromeres; Group 21-22, very short acrocentric chromosomes (includes the Y chromosome). These groups are often referred to by letters as A through G respectively. This international nonmenclature system is referred to as the Denver system. It is possible to identify individual chromosomes within a cell by use of an idiogram. They may be identified only by comparison with each other within a cell due to the varying degrees of contractions of chromosomes existing between cells (Robinson, 1961). This is accomplished by arranging paper cutouts of the chromosomes into pairs and then rearranging by size and centromere location until their classifications become clear. This method is very time consuming and involves much uncertainty. Recently, investigators have turned to a more precise method by using scanning computers to measure and calculate the relative lengths of the arms (Environment, 1970). This system of nonmenclature was further developed to describe variant chromosomes at a conference held in Chicago in 1966. They designated the short arm of a chromosome to be indicated by a p; the long arm by a q. Deletion from an arm of a chromosome by a minus sign, 6 and lengthening or an extra entire chromosome by a plus sign (German, 1970). A gene, being a region of a DNA molecule residing in the chromo- some, consists of purine & pyrimidine base pairs arranged in a Spec- ific sequence (code) which directs the manufacture of a particular enzyme or protein in the cell. The genetic code is altered by a Spon- taneous mutation rate at a level sufficient to permit rearrangments important to evolutionary development, but not so rapid as to exceed the ability of selective factors to eliminate deleterious mutations (Neel 3281111969). Agents capable of increasing this rate should be of concern when they are commonly introduced into the environment, since their effects will most likely be deleterious. When this genetic code is altered it may manifest itself in a viable genetic defect, neo- plasia or cell death. Damage to genetic material has been attributed to radiation, microorganisms and chemicals. Chromosome aberrations may involve either a numerical change or a structural change. Microscopically the effects of these substances can be observed in metaphase as aneuploidy, polyploidy, mitotic index, gaps, breaks, fragments, inversions, deletions, multicentrics, rings and translocations. Chromosome numerical changes include aneuploidy, polyploidy and mitotic index. Aneuploidy is any deviation in chromosome number from the normaldiploid state. It may be seen as a result of chromosome stickiness due to the action of some agent inhibiting movement during 7 division which causes irregular distribution to daughter cells. In some cases it may be an inherited trait seen either in all cells or in clones of cells. Also, aneuploidy arising as artefacts in slide preparation is found in cells of normal individuals. To determine whether the aneuploidy is random or due to a particular chromosome a karyotype analysis must be used. Aneuploidy is characteristic of rapidly dividing cancer cells (Environment, 1970). A polyploid cell is one which possesses two or more complete sets of chromosomes. It results from errors in division when the chromo- somes double, but the cell doesn't divide. This effect can be produced by a prolonged exposure to colchicine. Mitotic index is a comparision of dividing figures to non-dividing cells. It is included because it is a means of detecting effects of agents which may inhibit division. Chromosome structural changes are of two kinds, deletion and ex- change. The simple deletion is the result of a single break in the chromo- some or chromatid. The exchange is a new rearrangment following the joining of breakage ends which result from separate breaks. Break- age is the disruption of chromatin material which occurs before an aber- ration forms. If breaks do not restitute, they may lead to deletion, duplication, inversion or translocation of a chromosome. If a break occurs only in one arm of an already duplicated chromosome, i. e. in a single chromatid, it is a chromatid-type aberration; if both chromatids are altered, it is a chromosome-type aberration. Chromosome—type 8 aberrations (rings, multicentrics, exchanges, acentric fragments) are seen as a defect in both arms and are produced only in G0 or G1 when the chromosome acts as if it were functionally single stranded (Brewen, 1967). That is to say that the DNA double helix is the smallest unit in which an aberration will be seen. Little is known of the effect of partial breaks, i. e. subchromatid-type aberrations. However, they would most likely result in an unstable state which would either undergo restitution or further aberration. Of the possible chromosome-type aberrations only the trans- location would not result in an acentric fragment. An acentric fragment consists of two pieces of chromosome, with no centro- mere. If the lesion occured in a single chromosome an acentric fragment consisting of two pieces from the same chromosome could result. This lesion could be the result of one event affecting both strands of the chromosome or two events affecting both strands separately. If the lesion is a result of two events, one or two acentric fragments could result, depending on whether the fragments rejoin, prior to replication. A polycentric chromosome results from exchange between two (dicentric) or more chromosomes. It appears in metaphase as a single chromosome with more than one centromere and an acentric fragment. A ring chromosome results from an intrachange within a single chromosome. As the name suggests it is seen in inetanhase as a chromosome with its ends joined to form a ring and an acentric fragment. A trans- 9 location involves an exchange between chromosomes which, if equal and the position of the centromeres does not move, will not be seen. Usually, however, the exchange is unequal resulting in an unusually large chromosome. Generally a karyotype analysis is necessary to locate translocations. An exchange is an intra-change if it occurs within a single chromo- some or an inter-change if between chromosomes. The rearrangement is termed symmetrical (stable) if mitosis can occur without mechan- ical difficulty. An asymmetrical intra- or inter- change is one in which division is interfered with, as in the case of rings and multi- centrics. Chromatid-type aberrations are generally more stable than chromo- some-type aberrations, in that they are still capable of segregating properly at anaphase. A chromatid gap is a non-staining area less than one chromatid width and no terminal dislocation. If the stained portion of the chromatid distal to the gap is displaced or the non- staining area is greater than one chromatid width the aberration is a chromatid break. Gaps are usually considered insignificant because often they are artefacts resulting from poor stain uptake. An inversion occurs when a portion of the gene sequence becomes rearranged as a result of breakage and reunion in reverse order. A pericentric inversion includes the centromere, whereas a paracentric does not. Inversions are very difficult to detect except when they involve the centromere and the breakage points are not equidistant from it. In 10 this instance, for example, an acrocentric chromosome could be converted to a metacentric and would be detected in karyotype analysis. A fragment when listed separately from breaks indicates that a piece of a chromosome was observed but was not attributable to any part- icular chromosome. Similar to this is a deletion, which indicates that a chromatid arm is shorter than usual, but there is no evidence of a fragment. A defect in both chromatids of a chromosome and at the same level is an isochromatid aberration, which results in an acentric fragment. In human lymphocyte cultures both chromosome-type and chroma- tid-type aberrations could be found after the first in vitro division. Chromatid—type aberrations could result from damage in the lymphocyte mother cell or as a delayed effect of an agent affecting DNA synthesis during the in vitro division. Chromosome-type aberrations would be the result of an agent affecting the lymphocyte while it was in circulation since circulating human peripheral leucocytes are non-dividing. It should be noted that Bloom _e_t_a_l_._ in 1967 demonstrated clone formation in lymphocytes of atom bomb radiation exposed individuals. This would indicate that a small portion of the pOpulation may be actively dividing. The possibility exists that these cells are capable of dividing because of an aberration. The circulatory human peripheral leucocytes are in G013 v_i_\_r_<_)_ and only enter G1 when stimulatedE M(Bond _e_t El: , 1958; MacKinney _et 3L. , 1962; Bender and Prescott, 1962). Sasaki and Norman (1966) have shown that the small lymphocyte finishes its transformation in 24 11 to 48 hours after stimulation and begins a period of cell proliferation with a generation time of 22 hours; G1, S, G2 and M are 6,11, 3 and 2 hours, respectively. In a second paper, Sasaki and Norman (1967) showed that leucocytes may undergo up to three i__n _v_iLr_o divisions in 72 hours. Furthermore, they indicated that 70% of the cells ina 72 hour leucocyte culture system were in their second_i_n_v_it_13 division. This is contrary to the previously recognized paper of Bender and Prescott (1962) indicating that only one of seventy-five cells in a 72 hour leucocyte culture can be expected to be in second division. It is important to be sure that the cells only undergo one i_n_vi_t_5)_ division because the results will more closely resemble the conditions}; m A single leucocyte could be represented by four metaphase cells in 72 hours possibly giving an unrealistic percentage of stable aberrations. Buckton and Pike (1964) stated that the frequency of leucocytes with unstable type aberrations (dicentrics, rings and acentric fragments) decreases with increasing culture time. Also they showed that an increase in polyploidy occurs in leucocytes cultured greater than 72 hours. Contrary to this, Honda, Kamada and Bloom (1968) found no evidence of a decrease in aberrations with increased culture time. They compared the normal 66 to 72 hour culture with a 46 to 50 hour culture method of Bloom and Iida (1967). In 1969 Bender and Brewen offered an explanation which seemed to resolve the question. They found that a peak aberration rate occurred 54 hours after culture initiation which then dropped off with a second 12 peak, slightly smaller than the first, occurring at 78 hours. To account for this data they postulated that those leucocytes which divide in culture constitute at least two populations differing in the speed at which they reach mitosis. Furthermore they pointed out that not all leucocyte cultures reach second division at the same time. . The rate varies with different donors, incubation temperature and culture techniques. Since human circulatory leucocytes are essentially non—dividing (MacKinney gial_._,1962; Bender and Brewen, 1969), it is necessary to know the life of a leucocyte within the body to determine the time Span in which an effect could be seen. Aberrations seen_i_£1 v_i_t_rc_>_ are a con— servative estimate of damage Since severely damaged cells may die, there may be repair mechanisms operating to restitute damage or aberrations such as inversions and translocations may be undetected if the position of the centromere is not altered. The human leucocytes or white cells are of two basic types; those with granulated cytoplasm, granulocytes, and those without granules in the cytoplasm, agranulocytes. The agranulocytes include the small lymphocyte which merges through transition forms into the monocyte. The lymphocyte originates from precursor cells in the lymphatic tissue of the lymph nodes (Bloom and Fawcett, 1962) from which they are released into the peripheral blood. The leucocyte cells which divide in culture are the small lymphocytes (MacKinney galvl962). This imposes another technical limitation on aberration data in that of 13 the cells surviving the effects of an agent, whether lethal or inhibiting division, only that portion of the lymphocytes which are antigen-reSpon— sive (phytohemagglutinin) will enter mitosis. Buckton and Pike (1964) estimated the mean survival time of this circulating lymphocyte pop- ulation at several hundred days, a fraction of which may survive without division up to 10 years. Sasaki and Norman (1967) in their study of in- duced unstable aberrations reported a mean life of 530 days with some cells still existing without division at the end of the study, 2, 698 days. Bloom and Hamilton (1969) showed that heavily irradiated atom bomb survivors had a number of unstable aberrations attributable to the exposure 20 years earlier. Brewen (1970) indicated it is now generally agreed that small lymphocytes have a mean life of between 4 and 5 years. This average value probably represents a series of lymphocyte populations with different survival times. Removal of unstable aber- rations occurs in part when the lymphocytes enter division through antigenic stimuli and die as a result of the difficulties imposed by the aberration (Brown, 1967). As a result of the possible long life of lymphocytes, it becomes important to carefully check a subject's history for radiation therapy and other such mutagenic insults. Although the effects of agents on chromosomes can be described morphologically, relatively little is understOOd of the mechanism of chromosome breakage and repair at the molecular level. The bio- chemical lesions that lead to chromosome aberrations probably vary with different breaking agents. A lesion involves an alteration of the 14 in DNA. The alteration may be either submicroscopic, a point mutation, involving a small number of nucleotides or a gross aber- ration, such as a break which generally inhibits further DNA duplication. There are a variety of modes by which chemical agents can cause strand breakage. A chemical may inhibit DNA replication by blocking one of the mononucleotide precursors to DNA. The base analog 5-fluoro- deoxyuridine inhibits thymidylate synthetase, and, therefore, the con- version of deoxyuridylate to thymidylate is blocked (Taylor e_t_;_a_1_._, 1962). Another base analog, 5-bromodeoxyuridine, causes aberrations by incorporation into the DNA strand (Hsu and Somers, 1961). An agent may produce a chemical alteration of DNA. Ethylenimine is an alkylating agent and acts by facilitating depurination which has the effect of labilizing the sugar-phOSphate backbone (Chang and Elequin, 1967). Interference with repair of DNA lesions is another possible action. This mode of action has been proposed for caffeine (Cleaver, 1968). In contrast to enzyme inhibition, DNA damage could result from the enhancement of an enzyme such as deoxyribonuclease (Paton, 1970). Colchicine is a chemical which does not act by altering DNA, but rather disrupts cell division by inhibiting spindle formation. That aberrations could be induced artificially was demonstrated in 1928 by Muller with X-irradiation. Since then evidence of the health hazards of radiation has been extensive, including nuclear radiation, ultraviolet light and sunlight (Schrnickel, 1967; Buckton£t__a_l_.,l967; Bloom e_t_a_l_._, 1970; Bloom and Hamilton, 1969; Bloom 32.3.12 1966; 15 Bloom and Tijo 1964; Trosko 211211;, 1970). Recently, chromosome damage has even been attributed to viral infection (Nichols, 1966), specifically measles (Nichols _e_t_a_l_._,1962) and chicken pox (Aula, 1964). Chemicals comprise a larger group than radiation or micro- organisms and have been widely introduced into the environment. The first demonstrated chemical mutagen, mustard gas, was reported in 1947 by Auerbach, Robson and Carr. Known chemicals capable of inducing aberrations consist of many commonly occurring agents such as cyclamate (Stoltz 32%, 1970), caffeine (Ostertog, 1966), sodium nitrite (Malling, 1965), 3,4-benzpyrene (Epstein and Shafner, 1968), lysergic acid diethylamide (Egozwe__e_t_a_l. , 1968), mitomycin C (Shaw and Cohen, 1965), and captan (Legator e_t_a_._l_._, 1967'). These chemicals enter the body in such common forms as soft drinks, food, food ad- ditives, air and water pollutants, drugs, medications, and pesticides. Pesticides are of particular concern because their wideSpread use, dispersion and persistance in the environment makes contact with minute amounts unavoidable. These dosages, although usually too small to produce an acute effect. still may posess the potential for chronic effects. The majority of pesticides have not been adequately tested for mutagenicity in humans. The research that has been accomplished is inconclusive and generally only involves a test with one organism (Mrak Commission, 1969). Evidence of mutagenic activity exists for malic hydrazide (McCarthy and Epstein, 1968), captan (Legator _et_a_1_._, 1967 ), tretamine, TEPA and apholate (Chang l6 and Klassen, 1968; Epstein and Shafner, 1968) in human cells. Wuu and Grant (1966; 1967) have reported a systematic study of pesticides using barley root tips as have others in various systems (Mrak Commission, 1969). Although research in bioassay systems other than man is very informative, it cannot be said with certainty that the same Cause and effect relationship will exist in man. Distribution of the chemicals may differ greatly in human organs and tissues or they may be de- toxified by enzymes in human beings. On the other hand, compounds which are not mutagenic may be converted into mutagens in the human body. Because some pesticides are known to be mutagenic in lower organisms, a monitoring system must be employed to determine if man's exposure to these chemicals is detrimental enough to out-weigh the benefits. In humans the generation length makes it difficult to discover by direct observation that a chemical is producing mutations. A recessive mutation, if visible, would take at least two generations to be seen. Even a dominant mutation would not be seen until the next generation and by this time the effect, even if it were still possible to determine it, would have exposed large numbers. Therefore, it is important to detect a possibly carcinogenic, teratogenic or mutagenic agent early enough to rectify the cause. If the mutational effect were accompanied by chromosome breakage it could be detected by chromo- some studies in human leucocytes. MATERIALS AND METHODS The population studied was selected from high-use Sprayers of pesticides in the southwestern Michigan fruit-growing area. Dosage of the subjects was difficult to assess, even though accurate Spray records were kept, and could be determined only to number of days of usage. Furthermore, precautionary measures could not be stand- ardized. Control individuals were from the same area but had no direct contact with pesticides. The history of the subjects in the pre- ceding twelve months was checked for diagnostic X-irradiation ex- clusive of chest X-rays. In 1968 the average age of the exposed group was 43. 00 years and ranged from 24 years to 57 years. The control group had an average age of 46. 74 years and ranged from 30 years to 79 years. In 1969 the average age of the exposed group was 44. 74 years and ranged from 25 years to 58 years. The control group had an average age of 48.10 years and ranged from 32 years to 80 years. The study consisted of twenty control and twenty exposed male vol- unteers, examined each month during the Spray season. Three drops of whole blood, drawn by veinapuncture, were placed into a warm (37°C) culture tube of Grand Island Biological Company Chromosome Medium 1A and mixed well. The tubes were Shipped 17 18 via Special delivery mail in insulated containers to the laboratory. In l969 the technique was altered Slightly in that the blood was shipped in heparinized pipets and the cultures were initiated in the laboratory. The chromosome medium 1A contains culture fluid and fetal calf serum, heparin, antibotics, and phytohemagglutinin. The peripheral leucocyte cells, exposed igvi_vo, were cultured i_n_vfl:_i_‘_o at 370C for 72 hours. Three hours prior to harvest, 0. 3 milliliter Colcemide (10 micrograms per milliliter) in isotonic saline was mixed into each culture. At 72 hours the culture was transfered to a 12 m1. centrifuge tube and spun down at approximately 500 RPM for 3 minutes. The supernatant was removed and three milliliters of one percent sodium citrate, warmed to 37°C, was used as a hypotonic solution to resuspend the cells. After ten minutes the cells were recentrifuged at approximately 500 RPM for 3 minutes and with a clinical centrifuge, all except a small portion of the supernatant was removed. The button of red and white cells was then mixed thoroughly. Two milliliters of freshly prepared cold Carnoy fixative (one part glacial acetic acid to three parts methanol) was added quickly and mixed thoroughly. Fresh Carnoy fixative was then added enough times to obtain a clear supernatant, resuspending the cells at each centrifugation (see above). Slides were either prepared at this time or the cells left in the fixative overnight. The cells were suspended in fixative a little more than the size of the button, prior to slide pre- paration. Clean slides were dipped in 70% methanol and two or three l9 drOps of the suspended cells were allowed to fall on the slide from a height of 6 to 8 inches. Immediately the Slide was passed through a flame to ignite the methanol. The water left on the Slide after the flame burned out was drained off. The slide was then examined under the microscope to determine if the cell concentration had allowed maximum spreading. If the Slide was not satisfactory, a second slide was made by either diluting or concentrating the cell suspension. The slides were either stained with aceto-orcein or Giemsa and made permanent with Permount . Apparently normal metaphase plates (Figure 1) were selected for analysis under low- power (ZOOX) magnification on the basis of their apparent intactness and on the quality of chromosome spreading. Once selected, the cell was included in the study. Aberrations scored were breaks (Figure 2), dicentrics, rings, translocations, fragments and deletions, as well as chromosome number. Gaps (Figure 3) were considered insignificant and not included in the data. Photomicrographs were taken using Kodak High Contrast Copy film and were printed on F-5 paper. All slides were number coded to reduce bias. The £21252 tissue culture experiments used human peripheral leucocytes and human amnion AV3 cells. Analytical standards of the pesticides were obtained from the Pesticide Repository, U. S. P. H. S. , Perrine, Florida. The peripheral leucocytes were innoculated in the laboratory from a single donor and incubated as previously described. The human amnion AV3 cells were plated into 60mm petri dishes in 20 Eagle's MEM medium (10% calf serum). The cells were exposed with pesticides during the final 12 hours of a 72 hour incubation period. The pesticide solutions were prepared in dimethyl sulfoxide not more than an hour prior to application. For the AV3 growth experiment the pesticide was added at time zero approximately three hours after plating. The AV3 cells were removed from the plates by means of a 5 minute contact with trypsin and then either fixed for chromosome analysis or counted in a hemacytometer. § 0 y'gc ' 4‘ B a; 3 M ‘1 a I " an: ”a? “a Cl. FIGURE 1. Normal human chromosome metaphase plate. 21 a human chromos ome. FIGURE 3. Gap in 23 RESULTS Rings, dicentrics and translocations were not observed during the two years of the study and only chromosome breaks with their associated fragments were recorded. In 1968 the total number of breaks in the user group was more than twice that of the control group (3. 9 percent vs. 1. 7 percent). This was not confirmed in 1969 where the user group was insignificantly higher than the control group (1. 7 percent vs. 1. 2 percent). In neither year did the amount of breaks prove to be statistically significant at the five percent level when tested on a fourfold contingency table. The number of breaks for the months of June and July in 1968 and July of 1969 were signié ficant at the five percent level. In both years there appears to be a slight rise in chromosome aberrations during the peak of the Spraying season. This was also noticed in the controls, but was more erratic and not as significant, (Table l). The chromosome number distribution for the exposed group during both years was within the range for the controls (Table 2). Table 3 indicates the wide variety of pesticides excountered by the user group during a typical Spraying season. However, in Figure 4 it can be seen that only a small number of pesticides were encountered 24 25 by the group in significant amounts. The wide range of acute toxi- cities of the pesticides used is illustrated in Table 4. Those pesticides which were encountered in large amounts were tested £11932 on human amnion AV3 cells and human peripheral leucocyte cells. A comparison was made of production of aberrations and inter- ference with growth between these and other pesticides of varying toxicity. Tables 5 and 6 show that in the leucocyte cultures captan at a concentration of l X 10'4M produced twelve percent breaks where- as only one other pesticide (carbaryl, two percent) produced any breaks at all. The Guthion culture produced the lowest number of diploid cells. Chromosome analysis of treated human amnion AV3 cells showed a percentage of aberrations within the normal range at a concentration of 1 X 10'4M. However, captan at a concentration ten times lower than the rest of the dosages (1 X lO‘SM) still had an aberration rate slightly above controls (Table 7). Growth studies indicated minimal reduction in cell growth after addition of pesticides at a concentration of 1 X 10'4M, with the exception of the captan treated culture which did not survive (Table 8). The graph of the data (Figure 5) clearly -5 demonstrates that captan at a concentration ofl X 10 M inhibited cell growth for up to 48 hours. 26 TABLE 1. Effect of pesticides on chromosome aberration. No. of cells with aberrations/ No. of cells analyzed Control breaks % Exposed breaks 1968 May 2/212 .9 3/310 June 2/178 1.1 13/332 July 3/100 3.0 25/336 August 7/325 2.2 7/275 Total lu/als 1.7 u8/1253 1969 June 9/258 1.6 10/616 July 2/597 .3 11/600 August 7/225 3.1 8/330 September 1/294 .3 6/997 October 5/229 2.2 5/298 Total 19/1603 1.2 90/2391 27 N.H m. m.om H.m m.H m. a m w 3mm km ma N can emmoaxm moms m. :. m.mm m.: m.H m. s m N has mm a m com Hopscoo .mme s. 0.0 3.5m. m.m a.“ m. a m o was so as m mos emmoaxm mama Ufioaaxflaom SEA 0: m: 3.2 33v mjnmo .02 saves coflsnfluuma uthsZ mEOm OEOQLU .LODESS OEOwOEOQLO CO mmUHOH#w0Q .%O #OOMMM . m mam/a. 28 TABLE 3. Distribution of exposed group to pesticides encounterd.1 Number of days exposed-during month. Pesticide April May June July August Captan 7 101 86 60 68 Carbaryl 0 H 16 90 32 Cyprex 66 80 9M 33 17 DDT 6 32 MB 10 8 Ferbam 0 25 35 21 3 Guthion 3 80 109 90 #5 HEOD 5 HS 31 7 0 Karathane 0 12 21 7 0 Lead Arsenate 0 22 29 21 2 Lime Sulfur 22 22 9 H 5 Oil 33 7 0 0 0 Parathion 7 NH 51 ' Al 20 Simazine 2 20 2 0 0 Sulfur 28 10” 98 51 22 TEPP 0 0 H 8 23 1 Other pesticides used less than twenty exposure days in any month: Acaralate, Alar, Amitrole, BHC, Imidan, Maneb, Napthaleneacetic acid, Paraquat, Phaltan, Phosphamidon, Phygon, Streptomycin, Systox, Thiodan, Zineb Z9 £395 7 c0330 1 amumao .1 8330 H 1‘ l a JUNE JULY AUG. .335m 7 xeumcno _ amumdu _ ~3st — asummD _ M soflfiso _ M x9230 .1 L an.» 1 I. Ne U R P _ _ . . _ L . t _ _ _ A O O O O O 9 8 7 6 5 :30 Izo~ IIO IOOF 4o 30 ommoaxm m>Hmc< COHpmswcmocoo epHoHpmem |>Hom mOHSHcmOHQ mHHmo mMCHm mo .02 coHvanspme SonEoc mEOmoEopzo .OLHH> CH mHHmo ww>ooosmH Hmseansmm seas: mo mmEomoEosno co mmpHoHpmom mo Hoommm Ammgm<9 32 o m o NH o NH 0 3H 3 NH N ON N 3H N 0H N NH O NH om Nm mm mm mm 3m mm 3m 3m om mm oH (I): o NH CDC om om om om om om om om om om cm 2 IOmeN.H H Hz -onH 3 z OHxH : 2 -onH .— 2 -onH +_ 2 -onH 3 f onH 3 z.-OHxH z.-onH : -onH 3 Hospcoo omza QSMHsm mCHNMEHm GOHLHMme comm COansw xmhm>o Hmpmnsmo cepamo pHOHamHom pHOHmsmc< pHoHaHQ mama mxmmsm mCOHHMUOHmcmSB pemmHmc< moHspcmOHQ mmch Hzmommm m “HO HHmU .OZ COHHMchmocou mpHoHpmmm .ospH> CH mHHmo mp>ooosmH Hepmansem awed: mo meomoEosno co mmpHoHpmmm mo pommmm Awmqm appear in germ cells. Chromosome aberrations in human leucocyte cells can only serve as a warning indicator of a potential genetic effect in humans . SUMMARY An attempt was made to use human peripheral leucocytes as an Emmonitoring system of the subtle long term genetic effects of pesticides. The project was a pilot study to determine if a genetic risk existed. It demonstrated the feasibility of using human leuco- cytes, although the wide variety of pesticides used by the Sprayers made etiological conclusions difficult. Alterations of the test system were suggested to increase the validity of the data. The incidence of chromosome breakage in the exposed group was onlylslightly higher than that in the control group and larger numbers would be necessary to demonstrate a Significant increase. In general, assuming the adequacy of the test Systems used, the pesticides tested i_n_y}_t_r_g did not seem to present a human health danger from the gross cytological viewpoint. However, captan was demonstrated to be of concern due to its increase of chromosome aberration rate and inhibition of cell growth. 39 2) 5) 7) Recommendations for future pesticide-chromosome research Monitor larger numbers of subjects on a once a year basis due to the long mean life of lymphocytes. Attempt to establish a formulator group, contacting only a Specific pesticide, in addition to the Sprayer group. Screen all pesticides encountered in an_i__r_1_ vflgtissue culture situation. Investigate the use of an intraperitoneal diffusion chamber in ex- perimental mice to have a controlled and more realistic screening system. Count all cells analyzed Collect and culture blood using the methods described by Katz §_t_a_l_., 1970. Initiate two cultures per subject and harvest at 55 and 65 hours to assure Significant mitotic plates. 40 ' V n In“ I"! . I BIBLIOGRAPHY BIBLIOGRAPHY Auerbach, C., Robson, J. M., and Carr, J.C. 1947. The chemical production of mutations. Science. 105: 243-247. 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