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.     

Utero-ovarian infection in aged B6C3F1 mice

An unusually high number of ovarian masses and cysts with purulent material were observed in the B6C3F1 mice on 2 year chemical carcinogenicity studies sponsored by the National Cancer Institute-National Toxicology Program. To determine possible etiology, some of these lesions were cultured for bacteria and a majority yielded Klebsiella sp. Necropsy records of 14,029 female mice in 91 chronic studies necropsied from 1979 to 1983 at six toxicology testing laboratories were reviewed to determine the incidence of lesions and distribution of this disease. Animals for these studies were obtained from barrier production colonies of six suppliers. The incidence of this lesion was low in animals less than 14 months of age, increased with age and reached a peak in 24-26 month old mice. Most animals having this lesion either died or were sacrificed in moribund condition, indicating that this is a life shortening disease of aged B6C3F1 mice. The incidence of lesions ranged from less than 1% to 70% in different chronic studies. There was a marked difference in the incidence in mice from different suppliers and the incidence rate was 2.6 to 15% depending on the source of the animals. The incidence of this lesion in some testing laboratories was several-fold higher than in others and ranged from 0.9 to 20%. The proportion of mice with this lesion was low in some laboratories irrespective of the source of the animals, whereas in other laboratories the incidence was several-fold higher with animals from some, but not all suppliers, indicating testing laboratory-supplier interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.     

Benzene increases protein-bound 3-nitrotyrosine in bone marrow of B6C3F1 mice

Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.     

Genotoxicity of TiO(2) anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO(2)-NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO(2)-NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day -1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg.kg TiO(2)-NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency of the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO(2)-NPs in the bone marrow. These results suggest that although TiO(2)-NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.     

Genotoxicity of furan in Big Blue rats

Furan is a multispecies liver carcinogen whose cancer mode of action (MOA) is unclear. A major metabolite of furan is a direct acting mutagen; however, it is not known if genotoxicity is a key step in the tumors that result from exposure to furan. In order to address this question, transgenic Big Blue rats were treated by gavage five times a week for 8 weeks with two concentrations of furan used in cancer bioassays (2 and 8mg/kg), and with two higher concentrations (16 and 30mg/kg). Peripheral blood samples taken 24h after the 5th dose (1 week of dosing) were used to assay for micronucleus (MN) frequency in normochromatic erythrocytes (NCEs) and reticulocytes (RETs), and Pig-a gene mutation in total red blood cells (RBCs). 24h after the last dose of the 8-week treatment schedule, the rats were euthanized, and their tissues were used to perform NCE and RET MN assays, the Pig-a RBC assay, Pig-a and Hprt lymphocyte gene mutation assays, the liver cII transgene mutation assay, and the liver Comet assay. The responses in the MN assays conducted at both sampling times, and all the gene mutation assays, were uniformly negative; however, the Comet assay was positive for the induction of liver DNA damage. As the positive responses in the Comet assay were seen only with doses in excess of the cancer bioassay doses, and at least one of these doses (30mg/kg) produced toxicity in the liver, the overall findings from the study are consistent with furan having a predominantly nongenotoxic MOA for cancer.     

Ankylosis of hock joints in group caged male B6C3F1 mice

Enlarged hock joints were observed during 1983 in B6C3F1 mice of chronic toxicity and carcinogenicity studies sponsored by the National Toxicology Program (NTP). Subsequently, approximately 9,500 B6C3F1 mice on 32 NTP chemical toxicity and carcinogenicity studies were evaluated for this condition by clinical examination. Group caged male B6C3F1 mice had thickening and reduced mobility of the hock joints at prevalences of 1.2% up to 6 months of age; 23% at 6 to 12 months of age; and 62% at 13 to 26 months of age. Group caged female B6C3F1 mice had a prevalence of 2% or less. Histologically, affected mice had periarticular exostoses on the bones of the hock joints, with formation of bony bridges around joints and deposition of new bone in joint spaces, resulting in partial or complete ankylosis. Individually caged male and female B6C3F1 mice were not affected. The cause of the ankylosis was not determined, but its occurrence in the NTP studies has been reduced by individual caging.     

Inhibition of macrophage accessory cell function in casein-treated B6C3F1 mice

Humoral immunity of casein-treated B6C3F1 mice was evaluated. Splenocytes from casein-treated mice sensitized in vitro exhibited a marked suppression in their antibody response to the T-dependent antigen, SRBC (82%) and T-independent antigen, DNP-Ficoll (80%). In contrast, a control response to the polyclonal antigen, lipopolysaccharide (LPS), was observed. Cell fractionation and crossover reconstitution assays of adherent (ADH) and nonadherent (NAD) splenocyte populations from vehicle and casein-treated mice indicated that: 1) ADH splenocytes from casein-treated mice were responsible for suppression of humoral responses, 2) NAD splenocytes from casein-treated mice reconstituted with vehicle or naive ADH cells abrogated the immunosuppression, and 3) suppression of humoral responses in cultures containing casein ADH splenocytes was due to this cell populations inability to function as accessory cells in humoral responses rather than induction of suppressor macrophages. Results from in vivo studies with casein-treated mice sensitized with sheep red blood cells or dinitrophenyl-Ficoll paralleled the in vitro results.     

Simultaneous determination of malachite green and its metabolite leucomalachite green in eel plasma using post-column oxidation

A rapid HPLC method with solid-phase extraction (SPE) clean-up for malachite green (MG) and leucomalachite green (LMG) in eel plasma was developed. MG and LMG were extracted with a buffered methanolic solution. The extract was subjected to aromatic sulphonic acid SPE. MG and LMG were eluted from the SPE column with methanol after a treatment with ammonia gas. The reconstituted eluate was analyzed on a Chromspher B column with acetonitrile-ion-pair buffer (ph 4.0) (6:4, v/v) as the mobile phase and detection at 610 nm after post column oxidation with PbO₂. The average recoveries for MG and LMG over the linear range of applicability (20–2500 ng/ml) were 82±1% and 83±1%, respectively. The limits of quantification were 5.0 μg/1 for MG and 0.9 μ/1 for LMG.     

Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography-ion trap mass spectrometry

A liquid chromatography-ion trap mass spectrometry method with three "time segments" has been developed to determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) in edible goldfish muscle. By using the optimized "time segments", MG and LMG as well as the internal standard atrazine-d(5) were analyzed with good sensitivity with positive ESI-MS in a single run. The homogenized fish muscle tissues were extracted with a solution of perchloric acid and acetonitrile, followed by partitioning with dichloromethane. Strata-x polymeric solid-phase extraction column was used for the clean-up process. The determination of MG and LMG was achieved by using a reversed-phase HPLC gradient program coupled with MS/MS in multiple-reaction-monitoring mode. Matrix calibration curves were linear over the ranges of 5-500 ng/ml for MG and 1-100 ng/ml for LMG. Recoveries of the fish tissue extraction at three spiked levels (2, 10 and 30 ng/g for MG as well as 0.4, 2 and 6 ng/g for LMG) were better than 71% and 89%, respectively. Relative standard derivations from six determinations were less than 8%. The method detection limits were 0.13 ng/g for MG and 0.06 ng/g for LMG.     

The role of Klebsiella oxytoca in utero-ovarian infection of B6C3F1 mice

A disease consisting of suppurative endometritis, salpingitis, perioophoritis and/or peritonitis has been an important problem in aging B6C3F1 mice on some chronic chemical carcinogenicity studies. Klebsiella oxytoca was identified as the most likely causative agent based on cultural isolations from lesions. A study was done to determine prevalence of K. oxytoca in the "normal" flora of mice from different breeding facilities. In a survey of 684 retired female breeder mice from 10 National Institutes of Environmental Health Sciences (NIEHS) and National Cancer Institute (NCI) production facilities, K. oxytoca was isolated from only 1% of nasopharynxes, vaginas and ceca in mice from 7 of 10 facilities. Epizootiology of the natural infection was investigated using the capsular and biochemical typing methods on 97 isolates of K. oxytoca from mice of 11 NIEHS and NCI production facilities and sentinel mice from three National Toxicology Program testing facilities. A few capsular types were associated with either lesions, nonlesion isolation sites, or certain facilities but the capsular typing method was not reproducible. No associations were found for any biotypes. A K. oxytoca isolate (capsular type 20, biotype A) from a typical case of perioophoritis was used in attempts to reproduce the natural disease in Klebsiella-free B6C3F1 female mice. Mice were inoculated at 6 months of age by the intravaginal, intrauterine or intraperitoneal route with one of four doses of K. oxytoca and killed at 4, 7 or 10 months post-infection. Some mice given high doses (10(6) or 10(8) colony forming units) of K. +oxytoca died of septicemia and a few developed mild inflammatory lesions in the uterus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carcinogenicity study of GSM and DCS wireless communication signals in B6C3F1 mice

The purpose of this study using a total of 1170 B6C3F1 mice was to detect and evaluate possible carcinogenic effects in mice exposed to radio-frequency-radiation (RFR) from Global System for Mobile Communication (GSM) and Digital Personal Communications System (DCS) handsets as emitted by handsets operating in the center of the communication band, that is, at 902 MHz (GSM) and 1747 MHz (DCS). Restrained mice were exposed for 2 h per day, 5 days per week over a period of 2 years to three different whole-body averaged specific absorption rate (SAR) levels of 0.4, 1.3, 4.0 mW/g bw (SAR), or were sham exposed. Regarding the organ-related tumor incidence, pairwise Fisher's test did not show any significant increase in the incidence of any particular tumor type in the RF exposed groups as compared to the sham exposed group. Interestingly, while the incidences of hepatocellular carcinomas were similar in EMF and sham exposed groups, in both studies the incidences of liver adenomas in males decreased with increasing dose levels; the incidences in the high dose groups were statistically significantly different from those in the sham exposed groups. Comparison to published tumor rates in untreated mice revealed that the observed tumor rates were within the range of historical control data. In conclusion, the present study produced no evidence that the exposure of male and female B6C3F1 mice to wireless GSM and DCS radio frequency signals at a whole body absorption rate of up to 4.0 W/kg resulted in any adverse health effect or had any cumulative influence on the incidence or severity of neoplastic and non-neoplastic background lesions, and thus the study did not provide any evidence of RF possessing a carcinogenic potential.     

Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis

The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two -globin chains (-1 and -2) and at least three β-globin chains, β-1, β-2 and β-3. This is one additional - and one additional β-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the -1, -2, β-2 and β-3 chains compared to the BALB/c mouse hemoglobins (, β^minor and β^major). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.     

High accuracy determination of malachite green and leucomalachite green in salmon tissue by exact matching isotope dilution mass spectrometry

A high accuracy method for the quantification of malachite green (MG) and leucomalachite green (LMG) in salmon is described. Analytical challenges including the effects of analyte instability and matrix suppression were minimised by the use of exact matching isotope dilution mass spectrometry. The developed method included overnight extraction in acidified acetonitrile/ammonium acetate buffer and analysis by LC-MS/MS utilising isotopic internal standards. This method was used to determine the level of MG and LMG in a sample of salmon used in an international inter-comparison organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The sum of MG and LMG was found to be 9.32+/-0.98ngg(-1) at the 95% confidence interval (relative expanded uncertainty 10.5% (k=2)). This encompassed the mean and median of the CCQM inter-comparison.     

Forestomach ulcers in Crj:B6C3 (C57BL/6NCrj x C3H/HeNCrj) F1 mice

An unusually high incidence of forestomach ulcers was observed in a mouse strain that is used frequently for long-term toxicology studies. Examination of 98 untreated male and 98 untreated female B6C3F1 hybrid mice, the majority of which were between 105 and 113 weeks of age, revealed forestomach ulcers in 52% of the males and 54% of the females. Glandular stomach ulcers were uncommon, being found in only four female mice. The incidence of the ulcers increased with age. The etiology of the lesion is unknown.     

Struvite urolithiasis in a B6C3F1 mouse.

In a 2 year carcinogenicity bioassay using B6C3F1 mice, one male mouse developed clinical signs near termination of the study, comprising skin sores around the prepuce, penile prolapse and urine scalding. The predominant finding at necropsy was a markedly distended urinary bladder filled with numerous crystallized particles. Microscopically, there was subacute cystitis with marked hyperplasia of the transitional epithelium. X-ray diffraction analysis of the crystals showed a diffraction pattern characteristic of struvite (ammonium magnesium phosphate). The implications of the spontaneous occurrence of bladder stones in rodents on long-term toxicology studies are discussed.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Carcinogenicity test in B6C3F1 mice after parental and prenatal exposure to 50 Hz magnetic fields

Some epidemiological studies suggest association of childhood cancer with occupational exposure of the parents to magnetic fields. To test this relationship, 50 each of C57BL/6J female and C3H/HeJ male mice were exposed for 2 and 9 weeks, respectively, to 50 Hz sham (group A), 0.5 (group B), and 5 mT (group C) sinusoidal alternating magnetic fields. They were mated under the exposure for up to 2 weeks, and the exposure was continued until parturition. All the B6C3F1 offspring, without adjusting numbers of animals, were clinically observed without exposure to magnetic field for a nominal 78 weeks from 6-8 weeks of age after weaning and then euthanized for pathological examination according to a routine carcinogenicity test. 540 pups entered the test, and the survival rate was 96.7%. No F1 mouse died of tumoral diseases before a male in A group died of stomach cancer at 43 weeks of age. The first animal death in the exposed groups due to tumor occurred at 71 weeks of age. Eighteen animals died before necropsy at 84-86 weeks of age. No significant difference was detected in the final number of survivors and incidence of tumors between groups A and B, or A and C. Concerning reproduction total implants in group B were less than in group A and the difference was on the borderline of significance (P=.05). This difference was not reproduced in a later duplicate experiment.     

Differential effects of low- and high-dose X-rays on N-ethyl-N-nitrosourea-induced mutagenesis in thymocytes of B6C3F1 gpt-delta mice

Carcinogenesis in humans is thought to result from exposure to numerous environmental factors. Little is known, however, about how these different factors work in combination to cause cancer. Because thymic lymphoma is a good model of research for combined exposure, we examined the occurrence of mutations in thymic DNA following exposure of B6C3F1 gpt-delta mice to both ionizing radiation and N-ethyl-N-nitrosourea (ENU). Mice were exposed weekly to whole body X-irradiation (0.2 or 1.0 Gy), ENU (200 ppm) in the drinking water, or X-irradiation followed by ENU treatment. Thereafter, genomic DNA was prepared from the thymus and the number and types of mutations in the reporter transgene gpt was determined. ENU exposure alone increased mutant frequency by 10-fold compared to untreated controls and over 80% of mutants had expanded clonally. X-irradiation alone, at either low or high dose, unexpectedly, reduced mutant frequency. Combined exposure to 0.2 Gy X-rays with ENU dramatically decreased mutant frequency, specifically G:C to A:T and A:T to T:A mutations, compared to ENU treatment alone. In contrast, 1.0 Gy X-rays enhanced mutant frequency by about 30-fold and appeared to accelerate clonal expansion of mutated cells. In conclusion, repeated irradiation with 0.2 Gy X-rays not only reduced background mutation levels, but also suppressed ENU-induced mutations and clonal expansion. In contrast, 1.0 Gy irradiation in combination with ENU accelerated clonal expansion of mutated cells. These results indicate that the mode of the combined mutagenic effect is dose dependent.