Carcinogenicity of malachite green chloride and leucomalachite green in B6C3F1 mice and F344 rats.

Malachite green is a triphenylmethane dye used in the fish industry as an anti-fungal agent. Leucomalachite green is formed by the metabolic reduction of malachite green and persists in the tissues of exposed fish. In this study, we examined the carcinogenicity of malachite green chloride and leucomalachite green. Female F344 rats (48 per group) were fed diets containing 0, 100, 300, or 600 ppm malachite green chloride for 104 weeks, at which time the extent of tumorigenesis was assessed. Additional groups of 48 female and 48 male F344 rats were fed diets containing 0, 91, 272, or 543 ppm leucomalachite green for 104 weeks. Groups of 48 female B6C3F1 mice were fed diets containing 0, 100, 225, or 450 ppm malachite green chloride or 0, 91, 204, or 408 ppm leucomalachite green for 104 weeks. For each of the exposures, food consumption in the treatment groups was similar to the controls. Rats fed malachite green chloride or leucomalachite green had dose-dependent reductions in body weight; in mice, there were no consistent effects upon body weights with either compound. Female rats exposed to malachite green chloride had increased incidences of thyroid gland follicular cell adenoma or carcinoma and hepatocellular adenoma, and a dose-related increasing trend in mammary gland carcinoma. Female rats fed malachite green chloride and female and male rats fed leucomalachite green had a dose-related decreasing trend in the incidence of mononuclear cell leukemia. In male rats fed leucomalachite green there was a decreasing trend in pituitary gland adenoma and an increasing trend in interstitial cell adenoma of the testis. There were no treatment-related neoplasms in female B6C3F1 mice fed malachite green chloride. Female mice fed leucomalachite green had a dose-related increasing trend in the incidence of hepatocellular adenoma or carcinoma, with the incidence being significant in the highest dose group.

Suppression of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis by pre-initiation treatment of rats with beta-naphthoflavone coincides with decreased levels of the carcinogen-derived DNA adducts in the mammary gland.

BACKGROUND: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated. METHODS AND RESULTS: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt. for 4 days by oral gavage in corn oil before a single oral dose of DMBA (112 mg/kg b.wt.) suppressed mammary gland carcinogenesis as shown by an increase in the median latent period from 10 to 24 weeks and a 60% decrease in the multiplicity of mammary adenocarcinomas. In contrast, a 20-day treatment with beta-NF starting 3 weeks after DMBA had no significant effects on mammary tumorigenesis. The activities of phase I and phase II enzymes were examined in the liver and mammary gland 24 h after treatment of rats with beta-NF, DMBA, or beta-NF followed by DMBA as in the first bioassay. Treatment with either beta-NF or DMBA increased the hepatic activities of cytochrome P450 (CYP)1A1, 1A2, and 2B1/2, and glutathione S-transferase, and the mammary activity of CYP1A1. The activity of mammary CYP2B1/2 induced by DMBA was decreased by beta-NF. In the liver, the increase of UDP-glucuronosyl transferase (GT) activity in rats treated with beta-NF and DMBA was 2.3-fold greater than in rats treated with DMBA alone. Thus, treatment with beta-NF likely increased the rate of glucuronidation of DMBA dihydrodiols leading to carcinogen detoxification. The levels of the DMBA adducts determined by 32P-postlabeling of the mammary gland DNA were decreased in the beta-NF-pretreated rats. CONCLUSION: The beta-NF-induced increase in the hepatic UDP-GT activity and decrease in the mammary DNA-DMBA adducts occurred under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis.

High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts.

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

NTP technical report on the toxicity studies of malachite green chloride and leucomalachite green (CAS Nos. 569-64-2 and 129-73-7) administered in feed to F344/N rats and B6C3F1 mice.

Malachite green chloride is a triphenylmethane dye used in the fish and dye industries. Leucomalachite green is prepared by the reduction of malachite green chloride. Malachite green chloride was nominated for toxicity and carcinogenicity testing by the Food and Drug Administration and selected by the National Institutes of Environmental Health Sciences for carcinogenicity testing by the National Toxicology Program (NTP) due to the potential for significant worker and consumer exposure and lack of carcinogenicity data. The current 28-day studies were conducted as part of an overall effort by the NTP to determine the toxicity and carcinogenicity of malachite green chloride. Male and female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were exposed to malachite green chloride (95% pure) or leucomalachite green (99% pure) (male rats and female mice only) in feed for 28 days. Animals were evaluated for clinical pathology and histopathology. Genetic toxicity studies formalachite green chloride were conducted in vitro in Salmonella typhimurium and in vivo in rat bone marrow erythrocytes and in mouse peripheral blood erythrocytes. Genetic toxicity studies for leucomalachite green were conducted in vivo in mouse peripheral blood erythrocytes. Groups of eight male and eight female rats and mice were fed diets containing 0, 25, 100, 300, 600, or 1,200 ppm malachite green chloride for 28 days. Additional groups of eight male and eight female rats designated for thyroid hormone assays were fed diets containing 0 or 1,200 ppm malachite green chloride. Groups of eight male rats and eight female mice were fed diets containing 0, 290, 580, or 1,160 ppm leucomalachite green for 28 days. Additional groups of eight male rats designated for thyroid hormone assays were fed diets containing 0 or 1,160 ppm leucomalachite green. All rats and mice survived to the end of the studies. In the malachite green chloride study, the body weight gain of males rats in the 1,200 ppm group was significantly less than that of the controls. The final mean body weight of female rats and mice in the 1,200 ppm groups and the body weight gains of female rats and mice in the 600 (rats only) and 1,200 ppm groups were significantly less than those of the controls. In the leucomalachite green study, the final mean body weight of male rats and female mice in the 1,160 ppm groups and the mean body weight gains of male rats and female mice in the 580 and 1,160 ppm groups were significantly less than those of the control groups. In the malachite green chloride study, feed consumption by all exposed groups of male and female rats and mice was generally similar to that by the control groups. Exposure concentrations of 25, 100, 300, 600, and 1,200 ppm resulted in average daily doses of 3 to 190 mg malachite green chloride/kg body weight to male and female rats and 5 to 250 mg/kg to male and female mice. In the leucomalachite green study, feed consumption by all groups of exposed male rats was similar to that by the controls. Dietary concentrations of 290, 580, and 1,160 ppm resulted in average daily doses of approximately 30, 60, and 115 mg leucomalachite green/kg body weight to male rats and approximately 62, 110, and 220 mg/kg to female mice. In female rats exposed to malachite green chloride, there was a significant increases in gamma-glutamyltransferase activities with an activity in 1,200 ppm females seven times greater than that in the controls. Likewise, gamma-glutamyltransferase activity in male rats exposed to 1,160 ppm leucomalachite green was twice that in the controls. On days 4 and 21, the concentration of thyroxine was significantly decreased in male rats exposed to 1,160 ppm leucomalachite green and the concentration of thyroid-stimulating hormone was significantly increased. In the malachite green chloride study, the relative liver weights of 600 and 1,200 ppm male rats and the relative and absolute liver weights of 300 ppm or greater female rats were generally significantly greater than those of the controls. In the leucomalachite green study, the relative liver weights of 290 ppm or greater male rats were significantly greater than those of the control group. No gross lesions were observed in rats or mice and no microscopic lesions were observed in female mice that were attributed to malachite green chloride exposure. Microscopically, the incidences of hepatocyte cytoplasmic vacuolization were significantly increased in 1,200 ppm male and female rats exposed to malachite green chloride. No gross lesions were observed in rats or mice that could be attributed to leucomalachite green exposure. Microscopically, the incidences of hepatocyte cytoplasmic vacuolization were significantly increased in 580 and 1,160 ppm male rats. The incidence of multifocal apoptosis in the transitory epithelium of the urinary bladder was significantly increased in 1,160 ppm female mice exposed to leucomalachite green. Malachite green chloride, tested at concentrations of 0.1 to 10 mircrog/plate, was not mutagenic in any of several strains of Salmonella typhimurium, with or without S9 metabolic activation. Negative results were also obtained in two in vivo micronucleus tests, one that assessed induction of micronuclei in rat bone marrow erythrocytes after three intraperitoneal injections of malachite green chloride, and a second study that determined the level of micronuclei in circulating erythrocytes of male and female mice following 28 days of exposure to malachite green chloride via dosed feed. The frequency of micronucleated normochromatic erythrocytes in peripheral blood was significantly increased in female mice exposed to leucomalachite green in feed for 28 days; no significant increases in micronucleus frequencies were observed in the polychromatic erythrocyte population.

Synthesis and characterization of N-demethylated metabolites of malachite green and leucomalachite green.

Malachite green (MG), a triphenylmethane dye used to treat fungal and protozoan infections in fish, undergoes sequential oxidation to produce various N-demethylated derivatives (monodes-, dides(sym)-, dides(unsym)-, trides-, and tetrades-) both before and after reduction to leucomalachite green (LMG). The close structure resemblance of the metabolites with aromatic amine carcinogens implicates a potential genotoxicity from exposure to MG. The availability of the synthetic standards is important for metabolic and DNA adduct studies of MG. This paper describes a simple and versatile method for the synthesis of MG, LMG, and their N-demethylated metabolites. The synthesis involves a coupling of 4-(dimethylamino)benzophenone or 4-nitrobenzophenone with the aryllithium reagents derived from appropriately substituted 4-bromoaniline derivatives, followed by treatment with HCl in methanol. The resulting cationic MG and their leuco analogues showed systematic UV/vis spectral and tandem mass fragmentation patterns consistent with sequential N-demethylation. The extensive (1)H and (13)C spectral assignments of the metabolites were aided by the availability of (13)C(7)-labeled MG and LMG. The results indicate the existence of a resonance structure with the cationic charge located in the central methane carbon (C(7)). The synthetic procedure is general in scope so that it can be extended to the preparation of N-demethylated metabolites of other structurally related N-methylated triphenylmethane dyes.

The effect of deuterium and fluorine substitution upon the mutagenicity of N-hydroxy-2,6-dimethylaniline.

2,6-Dimethylaniline (2,6-DMA) is an intermediate in the manufacture of several products, including pesticides, dyestuffs, and synthetic resins. It is also present in nanogram amounts in tobacco smoke, and is a major metabolite of the potent anesthetic and antiarrhythmic drug lidocaine, as well as a nasal carcinogen in rats. As with other aromatic amines, 2,6-DMA can undergo metabolic activation through cytochrome p450-mediated N-hydroxylation, followed by O-esterification to a reactive derivative capable of forming DNA adducts. We have recently characterized four DNA adducts resulting from this metabolic pathway. Three of the adducts arose from reaction of the exocyclic heteroatoms of deoxyadenosine and deoxyguanosine with the carbon para to the arylamine nitrogen. The fourth adduct resulted from reaction of the 2,6-DMA nitrogen with the C8 atom of deoxyguanosine. In order to investigate the relative contribution of the exocyclic heteroatom adducts as compared to the C8-deoxyguanosine adduct to the toxicities elicited by 2,6-DMA, we synthesized and compared the mutagenicity of N-hydroxy-2,6-DMA, N-hydroxy-4-deutero-2,6-DMA, 2,6-dimethylnitrosobenzene, 4-deutero-2,6-dimethylnitrosobenzene, and N-hydroxy-4-fluoro-2,6-DMA. In Salmonella typhimurium TA100, the two deuterated compounds and their non-deuterated analogues gave similar mutagenic responses ( approximately 25 revertants/nmol). Likewise in S. typhimurium TA98, a similar mutant frequency ( approximately 0.7 revertants/nmol) was obtained with the four compounds. With N-hydroxy-4-fluoro-2,6-DMA, the mutant frequency was reduced by approximately 90% in S. typhimurium TA100 and approximately 50% in S. typhimurium TA98. The results suggest that multiple adducts contribute to base substitution mutations detected by S. typhimurium TA100 while the C8-deoxyguanosine adduct is primarily responsible for the frameshift mutations detected by S. typhimurium TA98.

Occurrence of H-ras codon 61 CAA to AAA mutation during mouse liver tumor progression.

The initiating mutations of a tumor are present in each of the cancerous cells comprising the tumor. Identification and measurement of the subsequent mutations that occur during tumor progression, however, requires mutation detection in a smaller subset of the tumor cells. In this study, allele-specific competitive blocker PCR (ACB-PCR), a genotypic selection method with the sensitivity to detect a specific point mutation in the presence of a 10(5)-fold excess of wild-type DNA sequence, was used to measure H-ras codon 61 CAA to AAA mutation in mouse liver tumors that did not have this mutation as an initiating event. Twenty-one spontaneous or chemically induced mouse liver tumors, negative for the H-ras codon 61 CAA to AAA mutation by DNA sequencing or denaturing gradient gel electrophoresis, were analyzed for this mutation by ACB-PCR. The mutation was detected at some level in 71% of these tumors. The mutation was detected in adenomas and carcinomas more frequently (13 of 14 tumors) and at significantly higher mutant fractions than it was detected in histiocytic sarcomas (1 of 5 tumors). These data indicate that the same oncogenic point mutation that can be identified as a tumor-initiating event based on its clonal amplification in a tumor can also be present in only a small sub-population of tumor cells where the mutation must have been fixed at a later stage in tumor development. The occurrence of a mutation as a primary or secondary event probably reflects the stochastic nature of mutation and is likely to be affected by the mutation rate for each target site.

DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver or mammary gland cytosol.

Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent mammary carcinogen in the rat. Potential adduct(s) of 2,7-diNF was (were) generated by reduction of 2-nitroso-7-NF with ascorbate/H(+) in the presence of calf thymus DNA. The major adduct was characterized by HPLC/ESI/MS and (1)H NMR spectrometry as N-(deoxyguanosin-8-yl)-2-amino-7-NF, and a minor one was determined by HPLC/ESI/MS to be a deoxyadenosine adduct of 2-amino-7-NF. Products from enzymatic nitroreduction were monitored by HPLC and DNA adduct formation by (32)P-postlabeling. Xanthine oxidase/hypoxanthine-catalyzed nitroreduction of 2,7-diNF, 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) yielded the respective amines to similar extents (30-50%). However, the level of the major adducts ( approximately 0.15/10(6) nucleotides) from 2-NF [N-(deoxyguanosin-8-yl)-2-aminofluorene] and 2,7-diNF [N-(deoxyguanosin-8-yl)-2-amino-7-NF] was < or = 2% that from 1-NP. In the presence of acetyl CoA, nitroreduction of 2-NF catalyzed by rat liver cytosol/NADH yielded the same adduct at a level of 2.2/10(6) nucleotides. Liver or mammary gland cytosol with acetyl CoA yielded mainly N-(deoxyguanosin-8-yl)-2-amino-7-NF from 2,7-diNF at >30 adducts/10(6) nucleotides, levels comparable to those from 1,6-dinitropyrene and 4- or 49-fold greater than the respective levels without acetyl CoA. Recovery of 2-nitroso-7-NF and 2-amino-7-NF from cytosol-catalyzed reduction of 2,7-diNF indicated nitroreduction and an N-hydroxy arylamine intermediate. Likewise, the presence of 2-acetylamino-7-NF indicated that reactivity with acyltransferase(s) was not prevented by the nitro group at C7. These data are consistent with activation of 2,7-diNF via nitroreduction to the N-hydroxy arylamine and acetyl CoA-dependent O-acetylation of the latter to bind to DNA. Enzymatic nitroreduction of 2,7-diNF was greatly enhanced by 9-oxidation. The nitroreduction of either 9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF catalyzed by liver cytosol with acetyl CoA yielded two adducts (>2/10(6) nucleotides). Differences in the TLC migration of these adducts, compared to those from 2,7-diNF, and the lack of 2,7-diNF formation in the incubations suggested retention of the C9-oxidized groups. The relative ratios of the amine to amide from nitroreductions of 9-oxo-2,7-diNF and 2,7-diNF catalyzed by liver cytosol suggested that the 9-oxo group decreased reactivity with acyltransferase and, thus, the amount of N-acetoxy arylamine that binds to DNA. The mammary gland tumorigenicity of 2,7-diNF and the extent of its activation by the tumor target tissue shown herein suggest relevance of this environmental pollutant for breast cancer.

Characterization of DNA adducts derived from syn-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide and comparative DNA binding studies with structurally-related anti-diolepoxides of benzo[ghi]fluoranthene and benzo[c]phenanthrene.

This paper reports structural characterization of the adducts and tetraols formed from syn-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide (syn-B[ghi]FDE, 3) and comparative DNA-binding and mutagenicity studies involving 3, anti-B[ghi]FDE (2), and anti-benzo[c]phenanthrene-11,12-dihydrodiol-13,14-epoxide (anti-BcPDE, 5). The structures of nine DNA adducts and two racemic tetraols derived from 3 have been determined spectroscopically. Similar characterization of adducts obtained from the anti-isomer 2 was described in the preceding paper in this issue [Chang et al. (2002) Chem. Res. Toxicol. 15, 187-197]. The majority of DNA adducts with 3 are those from the trans- or cis-opening of the epoxide at C5a by the exocyclic amino groups of dG, dA, and dC. The diolepoxides 2 and 3 are rigid structure analogues of anti- and syn-BcPDE (5 and 6), respectively, thus serving as models for probing molecular deformity and diol conformation in diolepoxide-DNA interaction. Comparative DNA binding experiments indicate that 57% of 2 and 33% of 3 were converted into DNA adducts, whereas a 71% conversion was observed for 5. In general, lower percentages were observed with denatured calf-thymus DNA. As for base selectivity, 2 showed a greater affinity for dA relative to dG (dA/dG ratio, 0.79) than 3 (0.56) when reacted with native calf-thymus DNA. A much higher dA/dG ratio (1.41) was obtained for 5. The overall dA/dG ratios were lower with denatured DNA, indicating the importance of the secondary structure of DNA for both adduct formation and chemical selectivity. The T-shape pseudo-diaxial diols of 3 appears to have favorable electrostatic interactions with the nearby phosphate backbone in the minor groove of DNA, thereby yielding greater amounts of dG adducts than the pseudo-diequatorial 2. The anti-isomer 2 was found to be seven times more mutagenic than 3, but they are significantly less mutagenic than the nonplanar analogue 5 when tested in Salmonella typhimurium TA 100.