Sandra J. Culp

Malachite Green: A Toxicological Review

Malachite green, an N-methylated diaminotriphenylmethane dye, is used primarily as a therapeutic agent in aquaculture. In solution, the dye exists as a mixture of the cation (chromatic malachite green) and its carbinol base, with the ratio depending on the pH of the solution; the dye also can undergo chemical and metabolic reduction to a leuco derivative. Analysis offish tissue after exposure to malachite green indicates the presence of both chromatic and leuco forms, with the latter having a much longer tissue half-life. Malachite green intercalates with DNA, with a preference for A:T-rich regions, and the leuco derivative bears a structural resemblance to carcinogenic aromatic amines that can form covalent DNA adducts. Malachite green is mutagenic in Salmonella typhimurium TA98 in the presence of an exogenous metabolizing system. In mammalian cells, it shows marked cytotoxicity and the ability to induce cell transformation and lipid peroxidation. Results from carcinogenicity bioassays with malachite green have been equivocal; however, it appears to act as a tumor promoter, perhaps because of its ability to induce the formation of reactive oxygen species. These characteristics, plus its close structural similarity to carcinogenic triphenylmethane dyes (e.g., gentian violet) suggest that additional data are required to determine if human exposure to malachite green results in adverse health effects.

Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice

Malachite green, an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. Malachite green is reduced to and persists as leucomalachite green in the tissues of fish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm malachite green or 1160 ppm leucomalachite green for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of leucomalachite green. This was not observed with malachite green. Hepatocyte vacuolization was present in rats administered malachite green or leucomalachite green. Rats given leucomalachite green also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given leucomalachite green. A comparison of adverse effects suggests that exposure of rats or mice to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. N-Demethylated and N-oxidized malachite green and leucomalachite green metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats. 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that malachite green and leucomalachite green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to malachite green.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Cancer risk estimation for mixtures of coal tars and benzo(a)pyrene

Two-year chronic bioassays were conducted by using B6C3F1 female mice fed several concentrations of two different mixtures of coal tars from manufactured gas waste sites or benzo(a)pyrene (BaP). The purpose of the study was to obtain estimates of cancer potency of coal tar mixtures, by using conventional regulatory methods, for use in manufactured gas waste site remediation. A secondary purpose was to investigate the validity of using the concentration of a single potent carcinogen, in this case benzo(a)pyrene, to estimate the relative risk for a coal tar mixture. The study has shown that BaP dominates the cancer risk when its concentration is greater than 6,300 ppm in the coal tar mixture. In this case the most sensitive tissue site is the forestomach. Using low-dose linear extrapolation, the lifetime cancer risk for humans is estimated to be: Risk < 1.03 x 10(-4) (ppm coal tar in total diet) + 240 x 10(-4) (ppm BaP in total diet), based on forestomach tumors. If the BaP concentration in the coal tar mixture is less than 6,300 ppm, the more likely case, then lung tumors provide the largest estimated upper limit of risk, Risk < 2.55 x 10(-4) (ppm coal tar in total diet), with no contribution of BaP to lung tumors. The upper limit of the cancer potency (slope factor) for lifetime oral exposure to benzo(a)pyrene is 1.2 x 10(-3) per microgram per kg body weight per day from this Good Laboratory Practice (GLP) study compared with the current value of 7.3 x 10(-3) per microgram per kg body weight per day listed in the U.S. EPA Integrated Risk Information System.

Lymphocyte mutant frequency in relation to DNA adduct formation in rats treated with tumorigenic doses of the mammary gland carcinogen 7,12-dimethylbenz[a]anthracene

The ability of the rat lymphocyte hprt assay to detect tissue-specific carcinogens was evaluated using 7,12-dimethylbenz[a]anthracene (DMBA) administered under conditions that result in mammary gland tumors. Fifty-day-old female Sprague-Dawley rats were given single doses of 5 and 20 mg/kg DMBA by gavage, and the frequency of 6-thioguanine-resistant (TGr) T-lymphocytes was measured over a period of 21 weeks. A time- and dose-dependent increase in mutant frequency was found, with a maximum frequency found 9-15 weeks after treatment with 20 mg/kg of DMBA. Rats were also dosed with 1, 2.5, 5, 10, 15 and 20 mg/kg of DMBA and assayed for TGr mutant frequency 10 weeks after treatment. A significant linear dose-response was found, with all the DMBA doses resulting in significant increases in mutant frequency. To determine whether or not DMBA-induced mutants in rat lymphocytes reflected the DNA damage in the target tissue, rats were treated with 5 and 20 mg/kg of DMBA and spleen lymphocytes and mammary gland tissue were assayed for DNA adduct formation 1, 3 and 7 days later. A similar pattern of 32P- postlabeled adducts, involving both dG and dA nucleotides, was found in DNA from both the target tissue and the surrogate lymphocytes. Adduct formation was dose responsive in both tissues, with a 2.3- to 4-fold higher concentration in mammary gland as compared with lymphocytes. These results indicate that the rat lymphocyte hprt assay is sensitive to a mammary gland carcinogen and that similar types of DNA adducts are associated with both the lymphocyte mutants and the mammary gland tumors induced by DMBA.

Synthesis and Characterization of 4′-Amino and 4′-Nitro Derivatives of 4-N,N-Dimethylaminotriphenylmethane as Precursors for a Proximate Malachite Green Metabolite

Abstract This paper describes the preparation of 4′-amino ( 2 ) and 4′-nitro ( 3 ) derivatives of 4- N , N -dimethylaminotriphenylmethane as precursors for presumed DNA-binding metabolites of malachite green. The primary amine 2 was synthesized via a condensation of 4-lithiated bis-( N , N -trimethylsilyl)aniline and 4-(dimethylamino)benzophenone. A simple nitro-dediazoniation reaction of 2 failed to afford 3 , but gave exclusively a mixture derived from hydro-dediazoniation followed by nitration and N -dealkylative- N -nitrosation. Direct nitration of N , N -dimethylaminotriphenylmethane, however, afforded 3 as well as other nitro isomers.

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.

DNA Adduct Measurements in Relation to Small Intestine and Forestomach Tumor Incidence during the Chronic Feeding of Coal TAR or Benzo[A]Pyrene to Mice

Abstract In a two-year carcinogenicity bioassay, female B6C3F1 mice were fed up to 100 ppm benzo[a]pyrene (BaP) or up to 1% coal tar in the diet. Forestomach tumors were induced in mice fed BaP, with the incidence increasing sharply between 5 and 25 ppm Bap. Forestomach tumors were also observed in mice fed coal tar, with a pronounced increase at the 0.3% dose. The incidence of forestomach tumors was approximately the same at 0.3% and 0.6% coal tar, but declined at the 1.0% dose, due to early mortality from a high incidence of small intestine adenocarcinomas. DNA adduct levels were examined in the forestomachs and small intestines after feeding BaP or coal tar for 4 weeks. In BaP-treated mice, one major adduct was observed; this adduct accounted for 7-15% of the forestomach adducts in mice fed coal tar. A dose-related increase was observed in adduct levels in the forestomachs of BaP-and coal tarfed mice. The induction of forestomach tumors from BaP or coal tar was associated with the same levels of BaP-DN...