Exogenous Metabolic Activation Research Articles

Mutation assays of ethyl methanesulphonate, benzidine and benzo[ a ]pyrene using Chinese hamster V79 cells

As a contribution to the third UKEMS collaborative trial, ethyl methanesulphonate (EMS), benzo[a]pyrene (B[a]P) and benzidine (BZD) were examined for mutagenicity at the hprt locus in Chinese hamster V79 cells, as assessed by 6-thioguanine (6TG) resistance. EMS was examined in the absence of any exogenous metabolic activation system, and the mutagenic dose-response obtained served to calibrate the assay. B[a]P and BZD were examined using three levels of Aroclor 1254-induced rat liver S9 supplemented with standardized concentrations of cofactors for NADPH generation. B[a]P showed S9 mediated cytotoxicity and mutagenicity, with the magnitude of both responses decreasing with increasing amounts of S9. No evidence of mutagenicity was seen for BZD with any of the metabolic activation conditions employed.

Chromosome aberration and sister chromatid exchange test results with 42 chemicals

Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (C.I. basic red 9 HCl, 2-mercaptobenzothiazole, oxytetracycline HCl, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79% reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.

Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro. V: Results With 46 Chemicals

Forty-six coded chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation. Sixteen chemicals were negative and 15 were positive in both assays; 15 were positive for SCEs only (one chemical that was positive for SCEs was equivocal for ABs), and no chemicals induced ABs only. The effect of cell harvest time on the ability to detect the induction of ABs was examined for 18 chemicals. Seven chemicals produced a positive response using both standard and extended harvest times, five were positive only using an extended harvest time, and six were negative using both harvest times. The relationship between cell cycle delay and SCE induction was also examined, and the two appear to be unrelated.

Evaluation of genotoxic effects of the herbicide dicamba using in vivo and in vitro test systems

The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S-9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.

Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S‐9 activation system

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.

Use of Frog Embryo Teratogenesis Assay-Xenopus and an Exogenous Metabolic Activation System to Evaluate the Developmental Toxicity of Diphenylhydantoin

Use of Frog Embryo Teratogenesis Assay-Xenoptis and an Exogenous Metabolic Activation System to Evaluate the Developmental Toxicity of Diphenylhydantoin. FORT, D. J. AND BANTLE, J. A. (1990). Fundam. Appl. Toxicol. 14,720–733. The teratogenic potential of diphenylhydantoin (DPH) and hydroxylated metabolites (HPPH) was evaluated with the Frog Embryo Teratogenesis Assay—Xenopus (FETAX). Embryos of the South African clawed frog, Xenopus laevis were exposed to DPH and HPPH in two separate static-renewal experiments with and without the presence of an exogenous metabolic activation system (MAS) for 96 hr. Two separate dose-response tests were also conducted with DPH and HPPH with a MAS modulated by various mixed functional oxidase inhibitors [carbon monoxide (CO) (broad spectrum cyto-chrome P450), cimetidine (mainly cytochrome P450), and ellipticine (cytochrome P448)] and an epoxide hydrolase inhibitor (cyclohexene oxide). Assessment of the potential teratogenic hazard was based on teratogenic indices [TI = 96-hr LC50/96-hr EC50 (malformation)], types and severity of malformations, and embryo growth endpoints. Addition of the intact MAS to DPH increased the 96-hr LC50 and EC50 (malformation) from approximately 74.5 and 32.4 mg/liter to 126.4 and 62.9 mg/liter, respectively. The TI was reduced 1.2-fold. Both p-HPPH and m-H PPH were much less developmentally toxic than DPH. CO and cimetidine inhibition of cytochrome P450 maintained much of the developmental toxicity of DPH, whereas ellipticine inhibition of cytochrome P448 was much less effective in maintaining the developmental toxicity of DPH. Cyclohexene oxide inhibition of epoxide hydrolase markedly increased DPH-in-duced embryotoxicity decreasing the 96-hr LC50 from approximately 74.5 to 38.6 mg/liter. These results suggest that unmetabolized DPH and an embryotoxic epoxide intermediate may serve as the teratogenic species in FETAX.

Evaluation of the developmental toxicity of five compounds with the frog embryo teratogenesis assay: Xenopus (FETAX) and a metabolic activation system.

The potential teratogenic hazard of five compounds was evaluated using the Frog Embryo Teratogenesis Assay--Xenopus (FETAX) and a metabolic activation system. Embryos of the South African clawed frog, Xenopus laevis, were exposed to (i) three compounds suspected to be proteratogenic in mammalian test systems--[2-acetylaminofluorene (2-AAF), rifampicin (RA) and benzo[a]pyrene (BP)] for 96 h; (ii) one compound unaffected by mixed-functional oxidase (MFO) metabolism--ZnSO4; (iii) one compound thought to be inactivated by cytochrome P-450--cytochalasin D (CD). Two separate static renewal tests were conducted with and without the presence of an exogenous metabolic activation system (MAS). The metabolic activation system consisted of Aroclor 1254-induced rat liver microsomes. The teratogenic potential of each compound and the effects of metabolic activation were based on teratogenic indices [TI = 96 h LC50/96 h EC50 (malformation)], types and severity of malformation, and effects on embryo growth. Metabolic activation increased the potential teratogenic hazard of 2-AAF, RA and BP by TI factors of 1.3, 2.8 and 6.8, respectively. The teratogenic potential of ZnSO4 was virtually unaffected by the MAS. The MAS significantly reduced the teratogenic potential of CD by a TI factor of 2.7. These results demonstrate the utility and importance of a MAS for in vitro developmental toxicity screens such as FETAX. Consistent use of a MAS with FETAX should reduce the number of potential false-positive and false-negative test results.

Influence of different factors on the induction of chromosome malsegregation in Saccharomyces cerevisiae D61.M by bavistan and assessment of its genotoxic property in the Ames test and in Saccharomyces cerevisiae D7

Bavistan is known to be a potent inducer of chromosome malsegregation in Saccharomyces cerevisiae. The influence of different factors on the induction of chromosome malsegregation in S. cerevisiae D61.M was investigated. With both standard protocols used (16 h overnight incubation and cold treatment protocol) bavistan, in a concentration range of 2.5–20 μg/ml, induced malsegregants to the same extent. The frequencies of malsegregants obtained were not influenced by the plating volume used on selective medium. Induction of malsegregants and toxicity became stronger with increasing supplementation of the incubation medium with yeast extract and peptone. The effects of bavistan on chromosome malsegregation were more pronounced at 28°C — the normal temperature for yeast growth — as compared to 33 and 37°C. A study of the time dependence of the induction of chromosome loss showed that malsegregants can already be detected after 8 h and 1.5 h (second incubation period) using the incubation protocols without and with cold treatment, respectively. To clarify whether a selection towards malsegregants occurs, the growth of mixed cultures of red, cycloheximide-sensitive cells and white, cycloheximide-resistant, leucine-auxotrophic cells prepared at different ratios was compared. A strong selection towards red cells and against the malsegregants was observed. In addition, bavistan was tested for genotoxic activity in Salmonella (Ames test) and in yeast S. cerevisiae D7. No mutagenic activity was detected using S. cerevisiae D7 (gene conversion, reverse mutation, mitotic crossing-over) with and without rat-liver S9. In contrast bavistan induced histidine revertants in the frameshift strains TA1537, TA1538, TA97 of Salmonella typhimurium after addition of an exogenous metabolic activation system.

Mutagenicity of tetrachloroethene in the ames test—metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of gamma-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the beta-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by beta-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.

Testing of sunset yellow and orange II for genotoxicity in different laboratory animal species.

The azo dyes Sunset Yellow and Orange II were gavaged to rodent species to check bile, urine, and fecal extracts for possible mutagenic activity in the Ames test or in bone marrow cells for clastogenicity using cytogenetic test systems. After oral application the dyes showed a negative response in bile, excrements, and bone marrow. When an exogenous metabolic activation was performed, increased revertant numbers using Salmonella strain TA100 were obtained only in fecal extracts of Sunset Yellow-treated animals. It is concluded that no genotoxic harm is to be expected from the ingestion of Sunset-Yellow or Orange II.

Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals.

The National Toxicology Program has undertaken a study to assess the ability of four genetic toxicology assays to predict the carcinogenicity of chemicals in 2-year rodent studies [Tennant et al.: Science 236:933-941, 1987]. Two of the assays, used for evaluating in vitro cytogenetic damage, were the SCE and chromosome aberration assays in Chinese hamster ovary cells. The results and data for 15 of the chemicals tested in these two assays are presented here. Each chemical was tested with and without exogenous metabolic activation. The chemicals tested were bisphenol A, 2-chloroethanol, C.I. acid orange 10, C.I. disperse yellow 3, C.I. solvent yellow 14, cytembena, D&C red 9, 1,2-dibromoethane, FD&C yellow 6, malaoxon, D,L-menthol, phenol, sulfisoxazole, titanium dioxide, and tris(2-ethylhexyl)phosphate. In vitro cytogenetic results from the other chemicals presented by Tennant et al. (Science 236:933-941, 1987) have been published by Galloway et al. (Environmental and Molecular Mutagenesis 10(Suppl 10): 1-175, 1987), Gulati et al. (Environmental and Molecular Mutagenesis 13:133-193, 1989), and Love-day et al. (Environmental Mutagenesis 13:60-94).

In vitro induction of chromosome damage by sulphasalazine in human lymphocytes

Two different endpoints, sister-chromatid exchange and micronucleus induction, were measured in human peripheral blood lymphocytes stimulated to divide in short-term in vitro cultures. The cultures were exposed to sulphasalazine and 6 of its metabolites for 72 h in the absence of any exogenous metabolic activation system. Analysis of the sister-chromatid exchange and micronuclei frequencies clearly indicates that sulphasalazine itself is capable of inducing both sister-chromatid exchange and micronuclei while sulphapyridine and its acetylated metabolites only induce sister-chromatid exchange. 5-Aminosalicylic acid, the therapeutic moiety of sulphasalazine, and its acetylated metabolite did not induce either sister-chromatid exchange or micronuclei at the concentrations tested. The data from these in vitro experiments are discussed in relation to the previously reported elevations in sister-chromatid exchange and micronucleus frequencies in inflammatory bowel disease patients receiving sulphasalazine therapy.

Evaluation of propanil and its N-oxidized derivatives for genotoxicity in the Salmonella typhimurium reversion, Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase, and rat hepatocyte/DNA repair assays

Since the herbicide propanil (3,4-dichloropropionanilide) is an aromatic amide and many other aromatic amides are genotoxic via N-hydroxy ( N-OH) metabolites, N-oxidized derivatives of propanil and 3,4-dichloroaniline were synthesized and tested for genotoxicity. Propanil, 3,4-dichloroaniline, and their N-OH derivatives were not mutagenic in the Salmonella typhimurium reversion assay using tester strains TA97, TA98, TA100, and TA104, in both the presence and absence of exogenous metabolic activation (S9). In addition, the test compounds were not mutagenic in the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay, in both the presence and absence of S9. 3,3′,4,4′-Tetrachloroazobenzene (TCAB) and its azoxy derivative (TCAOB), which are synthetic contaminants and/or degradation products of propanil, were also inactive in the S. typhimurium reversion and CHO/HGPRT assays (±S9). Unscheduled DNA synthesis (UDS) assays were performed to determine if propanil derivatives were able to induce DNA damage in primary rat hepatocytes. Although TCAB was the only derivative tested which induced an elevation in DNA repair, the extent was not statistically significant. Hepatocyte toxicity, as measured by the release of lactate dehydrogenase 24 hr after exposure, was induced by all the test compounds in a concentration-dependent manner. Incubations of [ 14 C]N-OH-3,4-dichloroaniline with DNA in vitro resulted in only a low level of binding that was not affected by pH. This observation may partially explain the lack of mutagenicity observed in genotoxicity assays with propanil derivatives.

Genotoxicity and PAC analysis of particulate and vapour phases of environmental tobacco smoke

Samples of indoor air were collected from an office room (88 m 3) both before smoking and during experimental smoking of 96 cigarettes by 10 persons within 6 h. The particulates were collected on glass-fibre filters and the vapour-phase compounds on XAD-2 resin. The samples were extracted with acetone and analysed quantitatively for polycyclic aromatic compounds and qualitatively with GC-MS. The extracts of filters and XAD-2 resins were fractionated into neutral/acidic and 2 basic (strong and weak bases) fractions; all these fractions were tested with the sister-chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells and with the Salmonella/microsome test (strain TA98). Total concentrations of PAC were 205 ng/m 3 in the background sample and 1207 ng/m 3 after contamination by cigarette smoking. The total PAC concentrations were 4–6 times higher in the vapour phase than in the particulate phase. The fractions of the particulate samples collected before smoking showed mainly marginal genotoxic activity, whereas after smoking their genotoxicity increased dramatically. The fractions of the vapour phase samples were not genotoxic before smoking, but after smoking the neutral/acidic and strong basic fractions induced responses in both assays. The SCE assay was more sensitive towards the vapour-phase mutagens of environmental tobacco smoke (ETS). The relative responses of the two bioassays were different: in the Ames test, the highest responses were obtained with the two basic fractions, whereas the fraction containing neutral and acidic compounds was the most potent in the SCE assay. In the Salmonella test, the mutagenic activity was mainly detected with metabolic activation, while the induction of SCE in CHO cells was also seen without an exogenous metabolic activation system.

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK +/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)- N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.

Inhibition of 7,12-dimethylbenz[a]anthracene-induced genotoxicity in Chinese hamster ovary cells by retinol and retinoic acid.

The effects of retinol and retinoic acid on cytotoxicity and mutation expression at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells with or without exogenous metabolic activation were studied in the presence and absence of known chemical mutagens. Neither retinol nor retinoic acid induced mutants at the HGPRT locus of CHO cells at concentrations ranging from 1 microM to 50 microM without exogenous metabolic activation, and at concentrations ranging from 1 microM to 25 microM in the presence of Aroclor 1254-induced rat liver S9. Retinol and retinoic acid did not affect 100 micrograms/ml ethyl methanesulfonate-induced cytotoxicity or mutations at the HGPRT locus of CHO cells in the absence of exogenous metabolic activation at concentrations ranging from 1 microM to 25 microM. In contrast, retinol and retinoic acid inhibited 7,12-dimethylbenz[a]anthracene-induced cytotoxicity and mutation induction at the HGPRT locus of CHO cells when either uninduced Sprague-Dawley rat liver S9, Aroclor 1254-induced Sprague-Dawley rat liver S9 or co-cultivated primary Sprague-Dawley rat hepatocytes were used to provide metabolic activation. These data show that retinoids are capable of inhibiting mutation induction in mammalian cells in vitro by a chemical promutagen requiring metabolic activation to a reactive form, and suggest that such inhibition is due to an alteration of mutagen metabolism.

Mutagenicity of hexachloro-1,3-butadiene and its S-conjugates in the Ames test--role of activation by the mercapturic acid pathway in its nephrocarcinogenicity.

The mutagenicity of hexachloro-1,3-butadiene and its S-conjugates 1-(glutathion-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GTB), 1,4-(bis-glutathion-S-yl-1,2,3,4-tetrachloro-1,3-butadiene (BGTB) and 1,4-(bis-cystein-S-yl)-1,2,3,4-tetrachloro-1,3-butadiene (BCTB) was investigated in Salmonella typhimurium TA100 using a modified preincubation assay. GTB was a direct-acting mutagen; the mutagenic potency of GTB was markedly enhanced by rat kidney microsomes or mitochondria and less so by cytosol. The bis-conjugates BGTB and BCTB were not mutagenic in the strains TA100, TA2638 and TA98. Purified HCBD was not mutagenic either without exogenous metabolic activation or with rat liver microsomes fortified with NADPH. Preincubation with rat liver microsomes and glutathione resulted in an unequivocal mutagenic activity of HCBD which was increased by additional inclusion of rat kidney microsomes. The cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased the mutagenicity of HCBD and its S-conjugates. These results provide strong evidence that formation of the corresponding monoglutathione S-conjugate from HCBD and subsequent cleavage of this conjugate by gamma-glutamyltranspeptidase and beta-lyase may be responsible for the nephrocarcinogenicity of the parent compound in vivo, whereas formation of the bis-glutathione S-conjugate probably plays no role in the organ specific effects of HCBD.

Evaluation of Propanil and Its N-Oxidized Derivatives for Genotoxicity in the Salmonella typhimurium Reversion, Chinese Hamster 1 Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase, and Rat Hepatocyte/DNA Repair Assays

Since the herbicide propanil (3,4-dichloropropionanilide) is an aromatic amide and many other aromatic amides are genotoxic via N-hydroxy (N-OH) metabolites, N-oxidized derivatives of propanil and 3,4-dichloroaniline were synthesized and tested for genotoxicity. Propanil 3,4-dichloroaniline, and their N-OH derivatives were not mutagenic in the Salmonella typhimurium reversion assay using tester strains TA97, TA98, TA100, and TA104, in both the presence and absence of exogenous metabolic activation (S9). In addition, the test compounds were not mutagenic in the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay, in both the presence and absence of S9. 3,3',4,4'-Tetrachloroazobenzene (TCAB) and its azoxy derivative (TCAOB), which are synthetic contaminants and/or degradation products of propanil, were also inactive in the S. typhimurium reversion and CHO/HGPRT assays (+/- S9). Unscheduled DNA synthesis (UDS) assays were performed to determine if propanil derivatives were able to induce DNA damage in primary rat hepatocytes. Although TCAB was the only derivative tested which induced an elevation in DNA repair, the extent was not statistically significant. Hepatocyte toxicity, as measured by the release of lactate dehydrogenase 24 hr after exposure, was induced by all the test compounds in a concentration-dependent manner. Incubations of [14C]N-OH-3,4-dichloroaniline with DNA in vitro resulted in only a low level of binding that was not affected by pH. This observation may partially explained the lack of mutagenicity observed in genotoxicity assays with propanil derivatives.

Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters.

Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M. This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII. The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment. The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9). Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin. Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments. In the case of the positive experiment, DES also induced putative recombinants. Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane). From our experiments it can be concluded that the hypothesis put forward by Parry et al. [Nature; 294:263-265], according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense. In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Clastogenic effects induced in mice and rats by 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene, a phenobarbital-like enzyme inducer and liver tumour promoter.

The clastogenic activity of 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), a phenobarbital-like enzyme inducer and liver tumour promoter, was studied in mammalian cells exposed in vivo and in vitro. The effects of an oral treatment with 3 mg/kg body weight of TCPOBOP were scored in the bone marrow cells and in the liver cells of B6C3F1 hybrid mice. A relevant increase in the frequency of chromosomal aberrations was observed in both cell types. A co-administration of TCPOBOP and carbon tetrachloride (the latter given to stimulate liver cell divisions) produced similar effects to those induced by TCPOBOP alone. Several i.p. doses (1, 3, 10, 30 or 100 mg/kg body weight) were administered to Swiss-albino mice and to Sprague-Dawley rats. In bone marrow cells, a remarkable concordance between the two species was observed: at any dose higher than 3 mg/kg body weight the increase in frequency of chromosomal aberrations was more than three times the control level, with a slight decrease at the highest dose. A dose of 300 mg/kg body weight was lethal. Doses of 3 or 30 mg/kg body weight were also administered i.p. to partially hepatectomized rats and the effects on metaphase chromosomes were detected in the liver cells. Besides an increase in structural chromosomal aberration frequency, TCPOBOP induced high percentages of hypodiploid and hyperdiploid-hypotetraploid liver cells. Since no changes in euploidy were observed in the bone marrow cells, this effect seems to be tissue specific. The clastogenic activity of TCPOBOP was also confirmed in vitro by a rat lymphocyte assay without addition of any exogenous metabolic activation.