Document 59gE5Xo6N8qVLMY2GOrQJwNR

t'amno^encsis Vo! 6 no 1 pp 69-72. !`-A> The rat liver foci bioassay: II. Investigations on the dose-dependent induction of ATPase-deficient foci by vinyl chloride at very low doses Reinhold J. Eaib1, Theresia Pellio2, Ursula M. Wiinschel2, Ncithard Zimmermann2 *an* d Hermann M. Bolt Institu; fur Arbeusphyriologie an der L!ni\e; Dortmund, Abteilung fur Toxikologie und ArbeiNmedizin, Ardeystr. 67, DM600 Dorimund 1, and *Phjrniakologisches Institut der Umversitat Main/, Obere Zahlbacher Sir. 67. D-650C Main/ 1, FRG `To whom reprim requests should be sent In order to study the dose-dependence of the genotoxie effect of vinyl chloride (VC) hepatocellular ATPase-deficient foci were evaluated after subchronie exposure of newborn rats. Wistar rats were exposed from day 1 after birth over 10 weeks to 10, 40, 70, 150, 500 and 2000 p.p.m. VC (8 h/day; 5 days/week). One week after cessation of exposure hepatic ATPase-deficient foci were quantitated. For a subsequent in vestigation lower dose range groups of female and male Wistar and Sprague-Dawley rats were exposed (8 h/day; 5 days/week) to 2.5, 5, 10, 20, 40 and 80 p.p.m. VC. Ex posure started at day 3 of life and lasted for 3 weeks. After cessation of exposure the animals were maintained for 10 weeks without further treatment until ATPase-deficient foci were quantitated. Both sets of experiments revealed a straight linear relationship between the dose of VC and the % foci area induced. Within the dose range investigated, no obvious threshold for the induction of pre-neoplastic foci by VC was observed. Introduction One major point of controversy in risk assessment of chemical carcinogens and in basic research on mechanisms of chemical carcinogenesis is that of `threshold' doses. Detoxifi cation of reactive metabolites and DNA-repair processes have been advoked to argue in favour of `thresholds'. On the other hand, DNA binding studies with established carcinogens so far have revealed linear dose-responses over wide dose ranges (1). In dose ranges covered by long-term bioassays the action of `genotoxie' carcinogens is found irreversible (persistent) and additive (2 -- 5). For statistical reasons in long-term animal experiments (20 -- 200 animals per group) tumor in cidences of 5 -- 10% over controls can be detected with statistical significance. Valid dose-response data at - 1% in cidence would require --500 animals per group; thus the animal number becomes a very limiting factor. Gehring and co-workers (6) have used combined bioassay (7) and metabolic (8) data on vinyl chloride (VC)* for risk prediction at low doses. They argued that a threshold for the carcinogenicity of VC might exist below exposure concentra tions of 50 p.p.m. because of the lacking depletion of hepatic glutathione in this dose range (8). The aim of the present study was to investigate the dosedependence of carcinogenicity of VC over an extended dose range, including very low exposure concentrations (2.5 *Abbreviations: VC, vinyl chloride; ATPasc, adenosine-5 '-triphosphatase. F IRl Press Ltd., Oxford, England. p.p.m.). As a sensitive parameter of carcinogenicity the development of pre-neoplastic enzyme-altered foci was in vestigated. Adenosinetriphosphatase (ATPase)-deficient foci were.quantitated as an endpoint of the `rat liver foci bio assay'. As foci area follows dose-time response relationships identical to that observed for the induction of liver tumors (4) this parameter was evaluated to describe the dose-response relationships observed. Materials and methods Animals Pregnant Wistar and Sprague-Dawley rats (Ivanovas Kissleg, FRG) were ob tained 7 -- 5 days before parturition and housed singly. They received a stan dard pellet diet (Altromin 1320, Lage, FRG) and drinking water ad libitum. Protocols Exposure schedule A. Groups of 4-5 male and female newborn Wistar rats (randomly assigned from four individual litters to two different exposure groups) were exposed together with the mother animals, starling from day 1 after birth for 10 weeks (S h/day, 5 days/week) to 10, 40, 70, 150, 500 and 2000 p.p.m. VC. At the end of exposure the individual exposure groups were maintained for 1 week without further treatment until ATPaxe-defieient foci were quantitated. Exposure schedule B (see Figure I). Newborn male and female Wistar and Sprague-Dawley rats were randomly assigned from four individual litters to two different exposure groups and exposed to VC together with the mother animals Exposure started at day 3 of life and lasted for 3 weeks (S h day; 5 days/week). Exposure concentrations were 2.5, 5, 10, 20, 40 and 80 p p.m. VC. At the end of exposure the animals were maintained for 10 weeks without further treatment until ATPase-deficient foci were quantified. Animals of each strain and sex which were exposed to air only served as controls. Exposure of the animals to VC (chemical purity 99^0, from Linde, Umerschleissheitn, FRG) was carried out as already described (9). Exposure concentration was routinely controlled by g.l.c. Exposure to high concentra tions of VC (2000, 500, 150 p.p.m.) was constant with daily deviat.ons of 51To of the intended value. At the lower concentrations, mean values for the 3 week exposure periods with maximal daily deviations were 80 * 11 p.p.m., 41 6 p.p.m., 20 5 p.p.m., 5.5 2 p.p.m. and 2.5 0.8 p.p.m. In the dose range where first order kinetics apply (10) the metabolized amount of VC (dose) is a function of the product of concentration and time and independent of the individual exposure profile. This implies that relatively Exposure Schedule Vinyl chloride i Sacrifice i no treatment ------ 1 nefwfbltoirn / Qo U oq*,,ikns 9,/ Wistar rats, Sprogoe-Dowley rats exposure to 0.2 5.5.10.20,40,BOppm VC number of onimals per exposure 7-99.7-9/ controls 219.161/ Fig. 1. Schcmaiic representaiion of the exposure schedule (schedule B) ap plied: exposure of 3-day-old Wistar and Sprague-Dawley rats of both sexes (S h/day; 5 day/week) to VC' for 3 weeks. After cessation of exposure the animals were maintained without further treatment until ATPase-deficient foci were quantified. 69 ASI 00004778 K.J I :iit> c. a! short deviations of the exposure concentration from a mean can be tolerated because the mean exposure concentration is or relevance only for such studies. HMochernxiry The animals uere sacrificed and cryostat sections front two Iiser lobes of each animal were prepared. The sections were stained for ATPase according to IWachstein el at. (I I). Foci area was quantitated in five cryostat sections per liver ( - 10 cm1) using the Zeiss lattice plate 11-100/25. Foci area was expressed as To of Iner area. The number of animals evaluated in each individual dose group is included in the legerd to the corresponding figures. Results for each exposure group are presented as means standard deviation. Dose-response curves are calculated b\ linear least squares fittings Results In a preliminary experiment groups of 4-5 male and female Wistar rats were exposed to concentrations between 10 and */t CngiosQfcomas fact area * * * Fig. 2. Incidence oT liver angiosarcomas (7) (top) and ATPase-deficient foci induced in female Wistar rats (hotlom) plotted versus exposure concentra tion (log/log scale) Incidence of angiosarcomas (6) and To foci atea in crease linearly with increasing exposure conctntra'ion until a plateau is reached. % foci orea 2000 p.p.m. VC according to schedule A and ATPasedeficient foci were quantitated in the livers of the animals 1 week after cessation of exposure. Figure 2 compares the influence on foci formation of saturation of VC metabolism with that on formation of angiosarcoma (7) evaluated by Gchring et al. (16). In female and male (data not shown) rats the foci areas show a linear in crease with increasing exposure concentration from 10 p.p.m. up to 500 p.p.m. VC where they reach a plateau of response. For a more detailed investigation in the lower dose ranee two strains of rats, a higher number of animals per individual dose group and a much higher number of controls was used. The animals were treated according to schedule B (Figure 1) and ATPase-deficient foci were quantitated 10 weeks after cessation of exposure (9). In Figures 3 and 4 foci area in the livers of male and female Wistar and Sprague-Dawley rats in dued by VC is plotted versus the different exposure concen trations. To demonstrate the large iiiterindividual differences in susceptibilities of the animals the standard deviations of the individual exposure groups are also included in the figures. (In this investigation the individual standard errors of estimate are ~ 1/3 of the standard deviations.) Figures 3 and 4 show that for male and female rats of both strains the foci area induced increases in proportion to the ex posure concentration (dose) applied. These dose-response relationships thus established within the range of 2.5 to 80 p.p.m. can be described, within the limits of error of the method, by linear dose-response curves which run through the origin. A comparison of the dose-dependent increase of foci area in both rat strains and sexes reveals increases in suscep tibilities in terms of induction of hepatocellular foci in the following order: ma'e Sprague-Dawley rats < male Wistar rats < female Sprague-Dawley rats < female Wistar rats. Also, it must be noted that mean foci area induced by the two lowest doses of VC in male rats of both strains lies within the range of foci area of the corresponding controls. Fig. 3. Do'C-dcpcndent induction of hepatocellular ATPasc-deficient foci by VC in male (left) and female (right) Wistar rats. The dove-response relationships can be described within the aectuacy of the method by linear dose-response curves which run through the origin. Number ol animals (in parenthesis) within the individual Jose groups examined for males: controls (14), 2.5 (8); 5 (6); 10 (7); 20 (5); 40 (10). SO (10); and lor females: controls (20), 2 5 (S); 5 (9): 10 (4); 20 (6); 40 (!2). SO (11) Dose-response relationship lor males is described by the linear curve > -- 7.4 x 10 4 + 0.32 y It)"3 \ wiih a coefficient of correlation of 0.86. and lot females y = 1.8 x 10 "4 + 0.5 x 10"3 ,x, with a coetfiuent of correlation of 0.R1 70 ASI 00004779 % foci oreo 1 Sprague-Dawley rats.d I Dose-dependent imi miion of A'lPnse-defkiem foci h> vinyl chloric % foci area (Sprogue-Dawley rots,?) Fig. 4. Dose-dependent induction of hepatocellular ATPase-deficient foci by VC in male (left) and female (right) Sprague-Dawley rats. The dose-response relationship can be described within the accuracy of the method by linear dose response curves which run through the origin. Number of animals (in paren thesis) within the indivdu.,1 dose groups examined lor males: controls 127); 2.5 (6); 5 (2); 10 (8); 20 (5); 40 (4); 80 (10); and for females: controls (10), 5 (15); 5 (7), 10 (9); 20 (7), 40 (7), 80 (I!). Dose-response relationship for males is described by the linear cune y = 3.5 x l(r< + 0.11 x ID-5 x with a coclticiem of correlation of 0.80 and for females y = 0,70 x 10"4 + 0 40 x 10" 3 x, with a coefficient of correlation of 0.91. Discussion The results of both studies (schedule A and schedule B) clear ly demonstrate a linear relationship between the dose (concentration) of VC applied and the area of ATPa.vedeficient foci induced, which ranges in doses over three orders of magnitude (2.5-500 p.p.m. VC). It is now clear that pharmacokinetics of a chemical have to be considered for risk evaluation (2,5). The risk of tumor development from VC is not directly related to exposure con centration but to the rate of metabolism of VC which is saturable at high exposure concentration (6). As VC metabolism follows first order kinetics until saturation occurs at high doses, tumor incidences increase linearly with increas ing exposure concentration until they reach a plateau at - 500 p.p.m. This has been demonstrated for angiosarcoma in cidence in rats by Gehring et si. (6) evaluating bioassay data on VC (7, see Figure 2). Our results show thai the induction of ATPase-deficient foci in female (see Figure 2, botiom) and male (results not shown) Wi.star rats is in accordance wfh these Findings. The dose-response relationships thus established for Wistar and Sprague-Dawley rats of both sexes in the lower dose region (2.5 -- 80 p.p.rn. VC) can be described by linear dose-response curves which run through the origin within the accuracy of the method. This exemplifies that within the dose range investigated no `threshold' dose exists for VC below' which the response prob ability is more than proportionally decreased. Earlier in vestigations on covalent binding of [14C]VC to cellular macromolecules are supportive of this view (12). In addition, the linearity of the dose-response curves disproves the hypothesis that a `threshold' for the carcinogenic action of VC in rats may exist at exposure concentrations below 50 p.p.m. (5, 8). These investigations also demonstrate that with the ap propriate protocol (9), the `rat liver foci bioassay' provides a very sensitive tool for detection of carcinogenicity. By quan titation of ATPase-deficient foci in the liver of rats, doseresponse relationships can be determined down to such low dose ranges where the carcinogenic risk caused by the substance can no longer be distinguished from that of nonexposed control animals. Acknowledgements The authors are grateful to J.Kaufmann for preparation of the histological slides, to D.Winter for typing the manuscript and to the `Deutsche Forschungsgemeinschatl' for financial support. References 1. Neumann.H.G. (19S0), Dose-response relationship in the primary lesions of strong electrophilic carcinogens. Arch. Toxicol. Suppl.. 3, 69-77. 2. Clemmescn.J., Conning,D.M., Henschler.D. and Oesch.F. (I9S0), Quan titative aspects of risk assessment in chemical carcinogenesis. Arch. Tox icol. Suppl 3. 3. Preussmann.R. (I9S0), The problem of thresholds in chemical car cinogenesis: some views of theoretical and practical aspects, J. Cancer Res. C/in. Oncol, 97, 1-14. 4. Kunz.H.VV., Tennekes.H.A., Port.R.E., Scbwarz,M, Lorke.D and Schaude.G. (1983), Quantitative aspects of chemical carcinogenesis and tumor promotion in liver, Environ. Health Perspect., 50, 113-122. 5. Schumann.A.M., Watanabe.P.G., Reitz.R.H. and Gehring,P.J. (1982). The importance of pharmacokinetic and macromolecular events as they relate to mechanisms of tumorigenicity and nvk assessment, in Plaa.G, and Hewitt,W'.R. (eds.), Toxicology of the Liver, Target Organ Tox icology Series, Raven Press, New York, pp. 311-331. 6. Gehring,P.J., Watanabe.P.G. and Park.C.N. (1978), Resolution of doseresponse toxicity data for chemicals requiring metabolic activation: exam ple - vinyl chloride, Toxicol. Appl. Pharmacol., 44, 581-591. 7. Maltoni.C, (1977), Recent findings on the carcinogenicity of chlorinated olefins. Environ. Health Perspect., 21, 1-5. 8. Watanabe.P.G., Zempel.J.A., Pegg.D.G. and Gehring.P.J. (1978), Hepatic macromolecular binding following exposure to vinyl chloride, Toxicol. Appl. Pharmacol,, 44, 571-579. 9. Laib.R.J., Klein,K.P. and Bolt.H.M. (1984), The rat liver foci bioassay: I. Age-dcpendcnce of induction by vinyl chloride of AT Pave deficient foci. Carcinogenesis, 5 (accompanying paper) 10 Fiber,.I.G. and Bolt.H.M. (1982), Pliarmahokineiische Aspckte von Arbcitxstoftypiuenhonzcntrationen, Verh. Dtsch. Ges. Arbensmed., 22, 631-635. ASI 00004780 71 K.J.Laib cl ii!. 11. Wadisiein.M, Meisd.E. anj Nied7wied/,A. (I960). Histodiemtc.il demonstration of mitochondrial adenosine triphosphatase with the leadadenosine triphosphate technique, J. flistochcm. CyKxhem , 8, 3S7-3SS. 12. Bolt.H M., Fiber,J.G., Laib.R.J. and Ottenwalder.H, (1980), Binding kinetics of virnl chloride and \in>l bromide at scry low doses, Arch To\icol. Suppl., 3, 129-U2 Received on 6 June 1984, accepted on 24 October 1984 72 ASI 00004781