Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.

Permeability, cytotoxicity, and genotoxicity of Cr(III) complexes and some Cr(V) analogues in V79 Chinese hamster lung cells.

The permeabilities and genotoxicities of the Cr(III) complexes [Cr(en)(3)](3+), mer-[Cr(glygly)(2)](-), cis-[Cr(phen)(2)(OH(2))(2)](3+), and trans-[Cr(salen)(OH(2))(2)](+) and the Cr(V) analogues of cis-[Cr(phen)(2)(OH(2))(2)](3+) and trans-[Cr(salen)(OH(2))(2)](+) [en being 1,2-ethanediamine, glygly being glycylglycine, phen being 1,10-phenanthroline, and salen being N,N'-ethylenebis(salicylideneiminato)] have been studied in V79 Chinese hamster lung cells. Following exposure of approximately 10(6) cells to 0.4 mM Cr(III) for 4 h, the Cr uptake by single cells was less than 10(-)(14) g/cell (as determined by GFAAS analysis and as confirmed by PIXE analysis where the Cr concentration was below the limit of detection). Importantly, the Cr(V) analogue of cis-[Cr(phen)(2)(OH(2))(2)] was significantly more permeable than the Cr(III) complex. The cytotoxicity of the Cr(III) complexes increased in the following order: mer-[Cr(glygly)(2)](-) < [Cr(en)(3)](3+) approximately cis-[Cr(phen)(2)(OH(2))(2)](3+) < trans-[Cr(salen)(OH(2))(2)](+). No genotoxic effects were observed following exposure to mer-[Cr(glygly)(2)](-) or [Cr(en)(3)](3+) at concentrations up to 6 mM. The Cr(III) imine complexes trans-[Cr(salen)(OH(2))(2)](+) and cis-[Cr(phen)(2)(OH(2))(2)](3+), which could be oxidized to Cr(V) complexes, induced MN in vitro at rates of 13.6 and 3.3 MN/1000 BN cells/micromol of Cr, respectively. The comparative permeabilities and genotoxicities of trans-[Cr(salen)(OH(2))(2)](+) and [CrO(salen)](+) were similar due to the instability of the Cr(V) complex at physiological pH values (7.4). There was a substantial increase in the permeability of [Cr(O)(2)(phen)(2)](+), compared to that of the Cr(III) analogue, which was accompanied by a highly genotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered as a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the genotoxicities of a variety of Cr(III) complexes when determining the carcinogenic potential of Cr(III) particularly when "high" deliberately administered doses are concerned.

MOCA and some proposed substitutes (Cyanacure, Conacure, Polacure 740M and Ethacure 300) as two-stage skin carcinogens in HRA/Skh hairless mice.

4,4'-Methylenebis(2-chlororaniline) (MOCA) is a suspect human carcinogen that has wide use as an industrial compound. Occupationally, exposure may occur through inhalation and ingestion, but skin absorption is the main route by which this compound gains entry into the body. Because of the justified concern about the continued use of MOCA, a number of substitutes have been proposed, including 1,2-bis(2-aminophenylthio)ethane (Cyanacure), Conacure, trimethylene glycol di-p-aminobenzoate (Polacure 740M) and 3,5-dimethylthio-2,4-toluenediamine/3,5-dimethylthio-2,6-tol uenediamine (Ethacure 300). There is very little information available about these substances, but they share the property of belonging to the same class (aromatic amines) as MOCA. Furthermore, at least two (Ethacure 300 and Cyanacure) are mutagenic in Salmonella. This study was undertaken to investigate if MOCA and substitutes, Polacure 740M, Ethacure 300, Cyanacure and Conacure have the potential to cause papillomas in a two stage initiation/promotion protocol in HRA/Skh hairless mice. When a maximum dose of 100 mg of substance was applied to the dorsal skin of these mice, Ethacure 300 and Cyanacure were markedly toxic. All of the compounds had little or no effect on skin tumor initiating activity following 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. One experiment with MOCA suggested that, at lower and less toxic dose, this substance may have promotional activity. Therefore, caution should still be exercised when using these compounds and it cannot be excluded that they may be active in other strains of mice or other laboratory animal species.

Permeability, cytotoxicity, and genotoxicity of chromium (V) and chromium (VI) complexes in V79 Chinese hamster lung cells.

The genotoxicity of Cr(V) complexes in mammalian cells (V79 Chinese hamster lung cells) has been studied for the first time using the in vitro micronucleus assay. Two complexes were investigated, [CrO(ehba)2]-, which undergoes ligand-exchange and disproportionation reactions in the cell growth medium, and [CrO(mampa)]-, which is chemically inert in the medium for the duration of the exposure period. Results of in vitro micronucleus assays show that both complexes are genotoxic and exhibit similar potencies to that of [Cr2O7]2-. The permeabilities of the Cr(V) complexes were also investigated for the first time using particle-induced X-ray emission (PIXE) analysis of individual cells. The Cr uptake increased in the order: [Cr(phen)2-(H2O)2]3+ < [CrO(ehba)2]- < [CrO(mampa)]- < [Cr2O7]2-. Clonal assays showed that Cr(VI) exhibits an expectedly higher cytotoxicity than the Cr(V) complexes. While the genotoxicities of the Cr(V) and Cr(VI) complexes increase according to their permeabilities, the genotoxicities of the Cr(V) complexes are equal to, if not greater than, that of Cr(VI) in terms of the amount of Cr entering the cell. This supports other evidence that Cr(V), produced as a metabolic intermediate from the intracellular reduction of Cr(VI), may be important in Cr-induced cancers.

Microprobe X-ray absorption spectroscopic determination of the oxidation state of intracellular chromium following exposure of V79 Chinese hamster lung cells to genotoxic chromium complexes.

The oxidation state of intracellular chromium has been determined directly in mammalian lung cells exposed to mutagenic and carcinogenic chromium compounds. Microprobe X-ray absorption spectroscopy (XAS) experiments on single V79 Chinese hamster lung cells showed that Cr(VI) and Cr(V) complexes were reduced completely (>90%) to Cr(III) within 4 h of exposure of the cells. This result provides direct evidence for the hypothesis that these genotoxic oxidants react rapidly with intracellular reductants.

[Metastasizing pleomorphic adenoma of the parotid. Apropos of a case]. / Adénome pléomorphe métastasiant de la parotide. A propos d'un cas.

A case of metastasizing pleomorphic adenoma of the parotid in which both the primary tumor and metastasis were composed of benign pleomorphic structures is reported in a 55 year-old white woman. The pulmonary metastasis developed nine years after excision of the primary tumor without local recurrence. No mitotic activity or infiltrative growth pattern were present in the primary tumor. By flow cytometry, DNA content was diploid in both the parotid tumor and the pulmonary metastasis. Previously reported cases are reviewed.

Evaluation of phosphine genotoxicity at occupational levels of exposure in New South Wales, Australia.

Phosphine has been claimed to cause chromosomal damage at exposures close to the current time weighted average exposure standard of 0.3 ppm (0.4 mg/m3). The current study involved 31 phosphine fumigators and 21 controls during the high fumigation season. All were volunteers and were evaluated for genotoxicity variables, including micronuclei in peripheral blood lymphocytes and urine mutagenicity. In parallel, all fumigators and 17 controls were evaluated for full haematology, multiple biochemical analysis, whole blood organochlorines, and whole blood and serum cholinesterase activity. The results for micronuclei showed no significant differences between fumigators and controls, but detected a strong association between age and increased frequency of micronuclei. Measurement of urine mutagenicity did not show any significant difference between fumigators and controls, but did show increased excretion of mutagens in smokers. All haematological and biochemical variables were within normal ranges, except for some non-specific changes in biochemistry. At monitored occupational exposures of < 2.4 ppm/h our results show no association between phosphine exposure and genotoxic or toxicological effects in fumigators.

Determination of genotoxic and other effects in mice following short term repeated-dose and subchronic inhalation exposure to phosphine.

Phosphine is an important fumigant in the grain industry and has been reported to be genotoxic in occupationally exposed fumigators. This study reports on the effects of phosphine inhalation exposure at up to, and exceeding, occupational relevant levels in a subchronic (0.3, 1.0 and 4.5 ppm, 13 weeks) and a short term repeated-dose (5.5 ppm, 2 weeks) study in both sexes of Balb-c mice. The following end-points were examined: micronucleus induction in bone marrow, peripheral blood, spleen lymphocytes and skin keratinocytes, mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in lymphocytes, and weight gain and relative organ weights (kidneys, lungs, liver, heart, brain and spleen). After subchronic exposure, there was a highly significant negative linear correlation between proportional weight gain and exposure in both sexes (multiple linear regression, r = -0.56, P < 0.0001), with female mice showing a greater effect. Females also showed an increase in relative organ weights at the highest test dose, in contrast to males where there was a slight decrease. A statistically significant increase in micronucleus frequency was seen in the bone marrow and spleen lymphocytes of both sexes, but only at the highest concentration. The short term repeated-dose study revealed a slight decrease in weight gain in both sexes, with a greater effect in females. It is concluded that phosphine is weakly genotoxic in both sexes of mice, and has an effect on weight gain. However, the weak genotoxic effect may not be biologically significant as it was seen only in the subchronic study and only at the highest test concentration of 4.5 +/- 0.8 ppm (approaching the LD50). Although such exposure conditions are unlikely to be encountered in an occupational environment, caution should continue to be exercised in the use of phosphine until more data become available.

Preparation, characterization, cytotoxicity, and mutagenicity of a pair of enantiomeric platinum(II) complexes with the potential to bind enantioselectively to DNA.

The synthesis of a pair of enantiomeric Pt(II) complexes, [Pt(R,R-eap)Cl2] and [Pt(S,S-eap)Cl2] (eap = N,N-diethyl-2,4-pentanediamine), designed to bind enantioselectively to GpG and ApG sequences of DNA is described. The in vitro cytotoxicity of each of the enantiomers toward murine leukemia and human bladder tumor cells has been measured. The R,R enantiomer was found to be more active in the leukemia cells, but the difference was not as great as expected (IC50; R,R 14 microM, S,S 33 microM). In the bladder tumor cell line, no significant difference in activity was found. The two enantiomers had similar mutagenicity in the Salmonella reversion assay, but the R,R enantiomer was more cytotoxic in the bacterial cells. A structural analysis of the R,R enantiomer revealed that the ligand adopted an unexpected configuration, and a strain energy minimization analysis showed that this was a consequence of interactions between the diamine ligand and the dichloro ligands. The significance of the structural preferences with respect to the lower than expected enantiospecificity is discussed. Crystals of [Pt(R,R-eap)Cl2] are monoclinic; space group, P2(1)2(1)2(1); a = 7.909(5), b = 12.972(9), and c = 13.269(12) A; Z = 4; and the structure was refined to R = 0.025 (1657F).

In vitro DNA damage and mutations induced by a macrocyclic tetraamide chromium(V) complex: implications for the role of Cr(V) peptide complexes in chromium-induced cancers.

Electron paramagnetic resonance and electronic absorption spectroscopies have shown that unlike the bidentate Cr(V) complex [Cr(ehba)2O]- (ehba = 2-hydroxy-2-ethylbutanoato(2-)), I, the macrocyclic tetradentate complex, [Cr (mampa-dcb)(O)]- (mampa-dcb = 5,6-(4,5-dichlorobenzo)-3,8,11,13-tetraoxo-2,2,9,9-tetrameth yl-12,12-diethyl-1, 4,7,10-tetraazacyclotridecane), II, is substitutionally inert. Low levels of DNA strand cleavage were observed after treatment with II under physiological conditions (50 mM sodium phosphate, pH 7.4, 37 degrees C) at concentrations as high as 2 mM for periods as long as 2 days. II also induces a lower number of revertants in mutation assays with Salmonella typhimurium TA100 than I when identical Cr concentrations are applied. The slopes of the linear portion of the dose-response curves are parallel, however, indicating that the mutagenicity of II is comparable to I. II is stable toward ligand exchange, reduction and disproportionation in the mutagenicity test medium and also in the presence of bacteria and the common cell reductant, glutathione. This indicates that ligand exchange with DNA and/or reduction to Cr(IV) are not responsible for the mutagenicity of II (unlike I). It is believed that II reversibly but weakly intercalates with DNA placing the Cr(V) center in close proximity for hydrogen atom abstraction or oxo-transfer reactions to ensure. This tetraamide complex is a good structural and biomimetic model for non-sulfur-containing Cr(V) peptide species that may form in vivo from reactions of Cr(VI) with peptides. Hence, it is likely to be relevant to understanding one possible mechanism by which Cr(VI) causes cancer.

Cytogenetic damage and tumor incidence in mouse skin after single, topical applications of 7,12-dimethylbenz[a]anthracene.

Micronucleus induction, chromosomal damage and aneuploidy were evaluated in whole skin keratinocyte cultures derived from HRA/Skh mice after single in vivo applications of 0.256, 2.56 and 25.6 micrograms (1, 10 and 100 nmoles) of the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). These genotoxicity end-points were compared with papilloma and carcinoma occurrence at the same dose levels of carcinogen. While the lower 2 doses of DMBA significantly increased the incidence of micronuclei and other chromosomal anomalies in keratinocytes, the two highest doses resulted in a significantly increased papilloma yield (0.297 and 3.895 papillomas/mouse) and incidence (24.3 and 100%). Carcinomas appeared only at the highest dose (0.125 carcinomas/mouse; 5% incidence). Neither papillomas nor carcinomas occurred in solvent-treated control mice. None of the three applied doses induced aneuploidy under conditions leading to an increase in tumors and/or chromosomal damage.

Evaluation of amitrole mutagenicity in Salmonella typhimurium using prostaglandin synthase activation.

Amitrole is a herbicide which has been found to induce thyroid and liver tumours in rodents, yet demonstrates limited genotoxic activity. The lack of mutagenicity of this compound in Salmonella typhimurium when employing a standard liver microsomal fraction, combined with evidence of activation of amitrole by peroxidases, warranted an investigation employing this other pathway of metabolic activation. Using prostaglandin H synthase as the activating system, the aromatic amine 2-aminofluorene provided a convenient positive control for optimisation of the metabolising system. Under such conditions, amitrole did not induce elevated numbers of revertant colonies in Salmonella typhimurium TA98, neither did it display evidence of interference with histidine biosynthesis as had been reported. Amitrole also remained nonmutagenic when preincubated at varying pHs. Thus, it has been shown that the alternative activation system, prostaglandin H synthase, does not produce metabolites which are mutagenic in the Ames test.

Skin carcinomas and micronucleus induction in epidermal keratinocytes following 12-O-tetradecanoylphorbol-13-acetate and mezerein treatment.

Phorbol esters are chemical compounds which have the ability to promote carcinogenesis in the skin of mice. In the HRA/Skh mouse strain, an initiation-promotion assay substantiated evidence that mezerein acts as a complete promoter. Complete promotion with 9.0 nmol mezerein induced a total of 53 papillomas in 28 mice within 40 weeks, while 1.5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA), using the same application regime, resulted in a total of 298 papillomas in 58 mice. However, 7.6% of the papillomas in the mezerein group progressed to carcinomas, compared with 0.7% in the TPA group. Approximately the same molar concentrations of TPA and mezerein induced a dose-dependent and reproducible increase in micronuclei in supra-basal cells derived from the epidermis of treated mice. In contrast, the latter compound was less toxic to these cells and induced higher numbers of micronuclei at the higher concentrations.

Tumorigenicity of cyclopenta[a]phenanthrene derivatives and micronucleus induction in mouse skin.

The most potent carcinogen of the cyclopenta[a]phenanthrene series, 15, 16-dihydro-11-methylcyclopenta[a]phenanthren-17-one and its non-carcinogenic, unmethylated parent compound, were compared for their abilities to induce micronuclei in epidermal keratinocytes after application onto the dorsal skin of Skh/HR-1 hairless mice. Although both substances were shown to be mutagenic in vitro, only the 11-methyl derivative has been proven to initiate cancer in TO and Sencar mouse strains. In the present study, only the 11-methyl derivative was active as a cancer initiator in Skh/HR-1 mice. For studying micronucleus induction, a preliminary experiment was conducted to establish doses of both chemicals that allowed cell survival. Subsequently, micronucleus induction in epidermal keratinocytes was shown to agree with the cancer-initiating potential of the two compounds. Only the carcinogenic derivative induced a statistically significant increase in micronuclei, over the range 10-100 nmol. This is considerably lower than the dose of approximately 1600 nmol commonly used to initiate skin cancer in mice, but is comparable to the active dose range for skin micronucleus induction by benzo[a]pyrene, a chemical of equivalent carcinogenic potency.

Micronuclei induction by dichlorvos in the mouse skin.

Micronucleus (MN) induction in cultured keratinocytes was investigated following skin painting of HRA/Skh mice with the pesticide, dichlorvos. Whole skin and partially-purified epidermal cells from 5-6 week old male animals were cultured for 4 days in vitro after single topical applications of various concentrations of dichlorvos in vivo. Appropriate doses, allowing optimum survival of keratinocytes, were selected following an initial range-finding experiment. To evaluate MN induction in dividing cells, the cytokinesis-block method was employed. Results showed statistically significant MN at all dose levels in partially-purified epidermal cells and a positive trend with respect to dose from 51 to 1033 nmol dichlorvos. A significant increase in MN was also detectable in cultured cells from whole skin, dissociated within as little as 1 h after application of dichlorvos. Although a number of technical difficulties are associated with the skin micronucleus method, it has been used successfully in this laboratory to detect several skin carcinogens of both high and low potency. Since dichlorvos is rapidly absorbed through the skin, and can induce MN in skin cells of treated mice, this compound may therefore be considered to pose a contact hazard for exposed humans.

Mutagenicity of basic fractions derived from lamb and beef cooked by common household methods.

Mutagen production was examined in lamb and beef in relation to certain common household cooking methods. Mutagenicity was assessed, after extraction of the basic fraction of cooked meat samples, using Salmonella typhimurium strain TA1538 with added rat-liver S-9 homogenate. Little or no mutagenicity was found in barbecued lamb chops, in microwave-cooked lamb chops, sirloin steak, leg of lamb, or rolled beef loaf, in roasted leg of lamb or rolled beef loaf, in stewed blade steak or in boiled chuck steak. However, the basic fraction from well-done, edible fried or grilled meat contained mutagenic activity equivalent to approximately 30,000 TA1538 revertants/100 g cooked meat. It was found tht the mutagenic activity of grilled lamb chops, sirloin and rump steaks was directly related to the average surface temperatures attained during cooking. Use of butter as a frying medium was particularly associated with higher mutagenicity in meat samples. Fried meats (rump and fillet steaks) generally yielded higher mutagenic activity than did grilled meats (rump steak, lamb chops) at comparable temperatures of the cooking medium. Using similar cooking procedures, lamb did not differ markedly from beef in mutagenic activity.

Genotoxicity and effects on rat liver drug-metabolizing enzymes by possible substitutes for 4,4'-methylene bis(2-chloroaniline).

The mutagenic properties of Ethacure 300, Cyanacure and Polacure 740M, all possible substitutes for the industrial carcinogen, 4,4'-methylene bis(2-chloroaniline), have been determined in Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537. These data have been compared with the effects of these chemicals on ethoxyresorufin O-deethylase (EROD) activity and aldrin epoxidase (AE) activity in rat liver. Ethacure 300 was clearly positive in both TA100 and TA98 bacterial strains, while Cyanacure was positive only in TA100. Polacure 740M was negative in all strains. Ethacure 300 caused a 28-fold induction of EROD while Cyanacure caused a doubling. Polacure 740M was without effect. Neither Ethacure 300 nor Cyanacure affected AE, while Polacure 740M caused an increase at only the lower dose tested. Thus there was excellent correlation between mutagenicity and EROD induction. A similar correlation was noted for six other structurally related compounds giving support to the contention that the ability of a chemical to induce EROD bears some relationship to its carcinogenic potential.

The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells.

Dibenz[a,j]acridine (DBAJAC) was studied because of its close structural relationship with a number of important carcinogenic polycyclic and azaaromatic hydrocarbons. It was of particular relevance to examine the mutagenicity of known or proposed 'bay-region' metabolites, which may be proximate or ultimate carcinogenic derivatives of DBAJAC. Trans-1,2-, 3,4- and 5,6-dihydrodiols, the 4- and 6-phenols, the 5,6-oxide and N-oxide derivatives, and anti- and syn-3,4-diol 1,2-epoxides of DBAJAC were examined for their mutagenicity in Salmonella typhimurium TA98 and TA100 and in V79 Chinese hamster lung cells. Of all the compounds studied which require metabolic activation, the 3,4-dihydrodiol was the most active in both TA100 and in V79 cells. The activity of the 3,4-dihydrodiol enantiomers was also tested in strain TA100 where no difference was observed from that of the racemic mixture. In V79 cells only the 3R,4R-dihydrodiol was active, the activity being about three times that of the racemic material. Salmonella strains TA98 and TA100 also differed in their sensitivity towards DBAJAC dihydrodiols, the 1,2-isomer being of greatest activity in TA98. The most mutagenic compounds in both mammalian and bacterial cells were the 'bay-region' diol epoxides of DBAJAC which did not require metabolic activation by S9 mix. The anti-DBAJAC 3,4-diol 1,2-epoxide was more mutagenic than the syn form in V79, TA98 and TA100 cells. Overall these results suggest that the in vivo biological activity of DBAJAC metabolites is likely to reflect previous findings with other similar polycyclic aromatic hydrocarbons.

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram.

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.