- Email: [email protected]
Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice
Chemico-Biological Interactions 122 (1999) 153 – 170
www.elsevier.com/locate/chembiont
Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice Sandra J. Culp a,*, Lonnie R. Blankenship a, Donna F. Kusewitt a,1, Daniel R. Doerge a, Louis T. Mulligan b, Frederick A. Beland a a
National Center for Toxicological Research, HFT-110, 3900 NCTR Road, Jefferson, AR 72079, USA b Center for Veterinary Medicine, FDA, Rock6ille, MD 20855, USA Received 24 March 1999; received in revised form 30 May 1999; accepted 3 June 1999
Abstract 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 metabo-
* Corresponding author. Tel.: + 1-870-543-7941; fax: + 1-870-543-7136. E-mail address: [email protected] (S.J. Culp) 1 Present address: University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. 0009-2797/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 1 9 - 2
154
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
lized 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. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Malachite green; Leucomalachite green; DNA adducts;
32
P-postlabeling; Apoptosis
1. Introduction Malachite green is an N-methylated diaminotriphenylmethane dye (Fig. 1) widely used in the fish and dye industries. The powerful antimicrobial activity of malachite green has been attributed to inhibition of intracellular enzymes, intercalation into DNA, and/or interaction with cellular membranes [1]. Although not approved for use in aquaculture in the USA, malachite green has been used as an antifungal treatment for fish since the 1930s [2]. Fish sold in the USA have not been routinely tested for contamination by malachite green; however, the compound is inexpensive, easy to obtain, and effective, which creates the potential for significant worker and consumer exposure. Random sampling of fish from markets in the UK indicated the continued use of malachite green in the aquaculture industry [3]. Data relating to the carcinogenicity of malachite green are extremely limited, although there is evidence of tumor promotion in rodent liver and suspicion of carcinogenicity based on structure–activity relationships (reviewed in Ref. [4]). Relatively little information is available concerning the metabolism of malachite green. Alderman and Clifton-Hadley [5] studied the uptake, distribution, and elimination of the dye after exposing trout to a 1.6 ppm bath treatment for 40 min. The maximum concentrations of malachite green in the serum, liver, and kidney (ranging from 7.8 to 34.0 ppm) occurred immediately after exposure, while a peak
Fig. 1. Structures of malachite green (MG), leucomalachite green (LMG), and demethylated derivatives.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
155
concentration (10.8 ppm) in the muscle was reached after 90–120 min. Other studies have shown that malachite green is reduced to and persists as leucomalachite green (Fig. 1) in the tissues of fish. For example, Law [6] showed a rapid absorption of malachite green in fingerling trout exposed to 2 ppm for 1 h. The malachite green in the whole fish homogenate decreased with time, while leucomalachite green increased up to 24 h after exposure and remained steady for the next 7 days. In this study, the effects of exposure to malachite green and leucomalachite green have been compared by feeding B6C3F1 mice and Fischer 344 (F344) rats the compounds for 28 days. Our data indicate that leucomalachite green causes a greater number of and more severe changes than malachite green. Examination of DNA and liver extracts indicates that the compounds may be metabolized in a manner similar to carcinogenic aromatic amines.
2. Materials and methods
2.1. Chemicals Malachite green (CAS registry number 569-64-2) and leucomalachite green (CAS registry number 129-73-7) were purchased from Chemsyn, Lenexa, KS. The chemicals were found to be ] 94 and ]98% pure, respectively, by high performance liquid chromatography (HPLC) with UV detection (254 nm) and evaporative light scattering detection, nuclear magnetic resonance spectrometry, atmospheric pressure chemical ionization mass spectrometry (APCI/MS), and elemental analyses. Impurities detected in malachite green were leucomalachite green (1%) and demethlyated derivatives of malachite green (3.5%). The impurities (5 2%) detected in leucomalachite green were monodesmethyl leucomalachite green and malachite green. (9)-Anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide was purchased from Chemsyn.
2.2. Animal treatments Female and male mice (eight per dose group per sex; B6C3F1/Nctr BR (C57BL/ 6N/X/C3H/HeN MTV-) as well as female and male rats (eight per dose group per sex; F344/N Nctr BR) were fed 0, 25, 100, 300, 600, or 1200 ppm malachite green for 28 days. Female mice and male rats (eight per dose group per sex) were fed 0, 290, 580, or 1160 ppm of leucomalachite green for 28 days. The animals were obtained from the breeding colony at the National Center for Toxicological Research, Jefferson, AR, and were 6–7 weeks old at the start of dosing. After the dosing period, blood was collected to measure clinical parameters, and tissues were examined histopathologically. Additional groups of male and female rats were fed 0 or 1200 ppm malachite green for 4 or 21 days (eight per dose group per sex per time point) and blood was collected for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) analyses. Male rats were treated likewise with
156
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
0 or 1160 ppm leucomalachite green (eight per dose group per time point). Another set of male rats and female mice (eight per dose group) was fed 0, 100, or 600 ppm malachite green or 0, 96, or 580 ppm leucomalachite green for 28 days and the livers were analyzed for metabolites and DNA adducts.
2.3. Necropsy and histopathology After 28 days of feeding, the animals were fasted overnight, but had free access to water. The animals were anesthetized with CO2 to collect blood for clinical chemistry measurements. They were then euthanized by CO2 exposure and a gross necropsy was performed. Tissues were fixed in neutral buffered formalin for 48 h, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin. Selected tissues were stained by ISEL (Apoptag kit, Oncor, Gaithersburg, MD), a technique to demonstrate DNA fragmentation. For thin sections, formalin-fixed tissues were embedded in glycol methacrylate, sectioned at 2 mm, and stained with toluidine blue.
2.4. Clinical chemistry Hematology measurements were conducted with a COBAS Minos Vet (Roche Diagnostic Systems, Branchburg, NJ) and included leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelet count, segmented neutrophils, lymphocytes, monocytes, eosinophils, and reticulocyte count. Clinical chemistry measurements were conducted with a COBAS Mira Plus (Roche Diagnostic Systems) and included total protein and bile acids, blood urea nitrogen, creatinine, alanine aminotransferase, and alkaline phosphatase. In rats, aspartate amino transferase, glucose, cholesterol, triglycerides, g-glutamyl transferase, albumin, sorbitol dehydrogenase, creatine kinase, sodium, potassium, chloride, calcium, and phosphorus were also measured. Total T3 and total T4 were determined with a ‘Coat-A-Count’ procedure obtained from DPC, Los Angeles, CA. The procedure is a solid-phase radioimmunoassay, wherein 125I-labeled T3/T4 competes with the sample for antibody sites. TSH was measured with a double antibody RIA procedure. Freshly prepared [125I]TSH was allowed to react overnight with the specific antibody and sample. An excess of second antibody containing polyethylene glycol was then added, bound and unbound [125I]hormone were separated by centrifugation, and the radioactivity was measured in the precipitates. The amount of sample was calculated from a standard curve.
2.5. Li6er extraction and analyses For HPLC analyses the livers were extracted using a method modified from Roybal et al. [7]. Specifically, 1 –10 g of liver was homogenized in a glass-Teflon homogenizer in a mixture of 2 ml 250 mg/ml hydroxylamine HCl in water, 3 ml 50
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
157
mM toluene sulfonic acid, 10 ml 100 mM ammonium acetate (pH 4.5), and 30 ml acetonitrile. Sodium chloride (2 g) was added and the samples were centrifuged for 10 min at 3000 rpm at 25°C. The supernatant was extracted with a mixture of 40 ml water, 2 ml diethylene glycol, and 90 ml methylene chloride. The organic layer was then concentrated to approximately 1 ml. Solid phase extraction was conducted as described in Roybal et al. [7]. The samples were analyzed by HPLC using a CN guard column, a 4.6 ×250 mm Spherisorb CN column, and a 20× 2.0 mm i.d. PbO2 oxidative post-column coupled to a Waters model 996 photodiode array detector. The following elution conditions were used with a flow rate of 1 ml/min: 0–10 min, 60% A, 40% B; 10 – 25 min a linear gradient to 50% A, 50% B (solvent A, 100 mM ammonium acetate (pH 4.5); solvent B, acetonitrile), and monitoring was conducted at 618 nm. The samples were quantified by comparison to malachite green or leucomalachite green standards. Untreated rat livers were spiked with the standards and processed in the same manner as treated livers to generate a calibration curve. No interconversion of the standards was observed in the spiked samples. The samples were also analyzed by HPLC in combination with APCI/MS. The separations were performed with a Dionex GP40 pump (Dionex, Sunnyvale, CA) and either a manual Rheodyne 7125 injector (Rheodyne, Cotati, CA) or a Dionex AS3500 autosampler. A Prodigy ODS-3 column (5 mm, 4.6× 250 mm) was used for separation of leucomalachite green and metabolites. The flow rate was 1.0 ml/min and a 10-min linear gradient was run from 50% acetonitrile in 50 mM ammonium acetate (pH 4.5), to 100% acetonitrile. Separation of malachite green and metabolites was performed using a Spherisorb S5 Nitrile column (80A, 5 mm, 4.6 × 250 mm) and a solvent system containing 40% acetonitrile in 50 mM ammonium acetate (pH 4.5), at a flow rate of 1.0 ml/min. For mass spectrometry, a VG Platform II single quadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with an APCI interface was used. The total HPLC column effluent was delivered into the ion source (150°C) through a heated nebulizer probe (500°C) using nitrogen as the probe and bath gas. Positive ions were acquired in full scan mode (m/z 100–800 in 1.5 s cycle time) and a UV detector set at 260 nm was placed in-line before the mass spectrometer. Two separate scan functions with different sampling cone-skimmer potentials were used to acquire mass spectra with minimal (20 V) or significant (60 V) amounts of fragmentation through in-source collision-induced dissociation. The desmethyl derivatives were synthesized (unpublished data) to confirm their presence in the samples subjected to APCI/MS. The samples were quantified by comparison to the response of the malachite and leucomalachite green standards.
2.6. DNA isolation and
32
P-postlabeling
DNA was isolated from the liver by the method reported in Culp and Beland [8]. Approximately 10 mg of DNA was 32P-postlabeled using n-butanol enrichment [9]. Adducts were separated by thin layer chromatography performed on 0.1 mm Machery Nagel 300 polyethylene imine cellulose plates (Alltech, Deerfield, IL) using the following solvent directions, D1: 0.9 M sodium phosphate (pH 6.8); D2: 3.6 M
158
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
lithium formate, 8.5 M urea (pH 3.5); D3: 1.2 M lithium chloride, 0.5 M Tris–HCl, 8 M urea (pH 8.0). A final wash was conducted in D3 with the solvent used in D1. DNA adducts were visualized using a Storm 860 phosphor imaging system (Molecular Dynamics, Sunnyvale, CA). The adduct levels were quantified by comparison to a 10b-(deoxyguanosin-N 2-yl)-7b,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene standard, obtained by reacting DNA with (9 )-anti-benzo[a]pyrene-trans7,8-dihydrodiol-9,10-epoxide.
2.7. Data analysis Body weights were measured weekly for a total of five measurements per animal and were analyzed on a per cage basis. A mixed-models approach to repeated measures analysis was used to model the mean cage body weights. The weights were modeled using the fixed effects of dose, time, and dose-by-time interaction. Dunnett’s two-sided test was used to test for differences between the control group means and each treatment group means. Contrasts were used to test for linear dose trends. A SAS GLM procedure was used to model the organ weights and the ratios of organ weights to body weights as functions of the dose. Dunnett’s test was used to test for differences between the control group mean and each treatment group mean. Clinical and hematology data were analyzed using a one-way analysis of variance (ANOVA) and Dunnett’s test. In instances where there was a non-normal distribution and/or an unequal variance, the analyses were conducted using a Kruskal– Wallis one-way ANOVA on ranks, with differences between the control group median and each treatment group median being tested by Dunnett’s method. DNA adduct levels were analyzed by one-way ANOVA, Dunnett’s test, and regression analysis. When an unequal variance occurred, the data were transformed by obtaining the square root before conducting the analysis Fisher’s exact test [10] was used to compare the proportion of lesions in the control group to that in each of the dosed groups. The Cochran–Armitage test [11] for a dose-response trend on proportions was used when all dose groups were examined. An exact test for this procedure was used because there were less than 10 animals in each dose group. All tests are one-sided in that decreases in incidence with an increasing dose were not considered. The P-values were adjusted with a modified Bonferroni procedure designed by Holm and modified by Wright [12]. 3. Results
3.1. Rats fed malachite green In female rats there were significant decreases in the mean body weights in the 1200 ppm dose group for weeks 1–4, with the animals weighing 80–83% of the control rats. Although the male rats fed 1200 ppm malachite green tended to have lower body weights (82 – 87%), as compared to the control group, the differences were not significant.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
159
Fig. 2. Liver to body weight ratios in (A) female and (B) male F344 rats fed malachite green or (C) male rats fed leucomalachite green for 28 days. Each data point is the mean 9 S.E.M. of eight animals. * Significantly (P B0.05) different from control group.
In the female rats, the animals in the three highest doses (300, 600, and 1200 ppm) of malachite green had significantly increased ratios of liver weights to body weights (Fig. 2A). The ratio of liver weight to body weight was significantly increased in the male rats fed 600 and 1200 ppm malachite green as compared to the control group (Fig. 2B). In both sexes, there was a significant linear increasing trend in the levels of g-glutamyl transferase, with the value in the 1200 ppm female dose group being 4.2-fold greater (P B0.0005) than that in the controls. Blood hematology measurements in female rats showed slight ( B7%), but significant, decreases in the 1200 ppm dose group in erythrocyte count, hemoglobin, hematocrit, mean erythrocyte hemoglobin, and mean erythrocyte concentration. Male rats had slight (B 3%), but significant, decreases in mean erythrocyte hemoglobin in the 300, 600, and 1200 ppm dose groups. Additional groups of female and male rats were fed 0 or 1200 ppm malachite green for 4 or 21 days and blood was collected for T3, T4, and TSH measurements. The T3 levels were significantly higher in female rats fed 1200 ppm malachite green as compared to the control group on day 21 (Table 1). The T4 levels were significantly lower on both days 4 and 21 in the female rats in the 1200 ppm group as compared to the respective control groups. There were no significant changes in T3 or T4 levels in males or in the TSH levels in either sex.
160
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Seven out of eight female rats fed 1200 ppm malachite green had minimal to mild hepatocyte vacuolization (P B0.01). The same lesion, primarily midzonal in location, was observed in one of eight male rats fed 600 ppm malachite green and four of eight male rats fed the 1200 ppm dose.
3.2. Male rats fed leucomalachite green Male rats fed 1160 ppm leucomalachite green had significantly lower body weights (91 – 92% of the control group) at weeks 2, 3, and 4. There were also Table 1 T3, T4, and TSH levels in female and male F344 rats fed malachite green or leucomalachite green for 4 or 21 daysa Clinical parameter
Compound
T3 (ng/dl)
Malachite green Leucomalachite green Malachite green
Time (days)
4
21
Leucomalachite green T4 (mg/dl)
Malachite green
4
21
Leucomalachite green TSH (ng/ml)
Malachite green
4
Leucomalachite green Malachite green Leucomalachite green
Dose (ppm) (mean 9S.E.M.)
Female Male
0 116.2 95.41 128.3 96.53
1200/1160 118.7 9 6.13 134.7 96.09
Male
113.7 9 5.25
108.0 94.35
Female Male
105.4 9 2.21 106.0 9 5.21
123.1 9 4.12b 111.1 97.60
97.5 9 3.56
98.5 94.06
Male
Leucomalachite green Malachite green
Sex
Female Male
3.1 9 0.14 3.8 9 0.17
2.6 90.14b 3.7 90.05
Male
5.0 9 0.29
3.4 90.18b
Female Male
3.0 9 0.14 3.5 9 0.05
2.5 90.11b 3.1 90.04
Male
3.0 9 0.17
2.3 90.13b
0.86 9 0.12 0.73 9 0.06
1.0 90.12 1.1 9 0.17
1.9 90.16
3.0 9 0.44b
Female Male Male
21
Female Male Male
0.75 9 0.09 1.2 90.15 3.7 90.80
0.97 9 0.13 1.2 9 0.17 6.3 9 1.58b
a Female and male F344 rats were fed 0 or 1200 ppm malachite green and male rats were fed 0 or 1160 ppm leucomalachite green for 4 or 21 days (eight per dose group per sex per time point). Blood was collected for T3, T4, and TSH analyses. The data are expressed as the mean9S.E.M. of 7–8 rats. b Significantly (PB0.05) different from control group at the same time point.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
161
significant decreases in body weights in the 580 ppm dose group at weeks 3 and 4 (94% of the control group). Linear decreasing dose trends were observed for weeks 2, 3, and 4 (P B0.003). The ratio of liver weights to body weights was significantly increased for all three dose groups as compared to the control group (Fig. 2C). g-Glutamyl transferase levels were 2.2-fold higher (PB 0.05) and phosphorus levels were slightly increased (10%; PB 0.05) in male rats fed 1160 ppm leucomalachite green. In addition, erythrocyte count, hemoglobin, and hematocrit levels showed slight (B6%), but significant, decreases from the controls in the 1160 ppm dose group. An additional group of male rats was fed 0 or 1160 ppm leucomalachite green for 4 or 21 days and blood was collected for T3, T4, and TSH measurements. There was a significant decrease in T4 and increase in TSH levels on days 4 and 21 as compared to the respective control groups (Table 1). In male rats fed leucomalachite green, hepatocyte vacuolization, primarily midzonal and centrilobular in location, was seen in 7/8 rats fed 1160 ppm (P B 0.005), 5/8 rats fed 580 ppm (P B0.04), and 2/8 rats fed 290 ppm, a significant dose trend (P B 0.0004). Two of the eight rats fed 1160 ppm and two of the eight rats fed 580 ppm leucomalachite green had apoptotic follicular epithelial cells in the thyroid gland. Morphologic changes consisted of sloughed follicular cells with condensed nuclei located within the follicles. An inflammatory reaction was not present. There was evidence of follicular epithelium regeneration, since even the most severely affected follicles were still lined by viable epithelium.
3.3. Mice fed malachite green Female mice fed 1200 ppm malachite green had significantly lower body weights (91– 92% of control group) at weeks 3 (PB 0.02) and 4 (PB 0.03). The body weights of male mice were not significantly effected by any of the dose levels of malachite green. In female mice fed 600 or 1200 ppm malachite green, there were slight (6–8%) significant decreases in the erythrocyte count and hemoglobin and hematocrit levels as compared to the control group; in male mice, significant decreases in these parameters were observed in the 1200 ppm dose group. The mean erythrocyte volume was increased 1 – 2% (PB 0.05) in female mice fed 300, 600, and 1200 ppm malachite green as compared to the control group. There was also a 1.4–1.9-fold increase in reticulocytes in these groups (PB 0.05). Male mice fed 1200 ppm malachite green showed a 1.6-fold increase in reticulocytes (P B 0.05). There were no significant histopathological changes observed in the mice fed malachite green.
3.4. Female mice fed leucomalachite green Female mice fed 1160 ppm leucomalachite green had significantly lower body weights (93% of the control group) at week 4. A marginally significant decrease (PB 0.01) from the control group was observed in the female mice fed 580 ppm at
162
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Fig. 3. 32P-Postlabeling scans of liver DNA from F344 rats or B6C3F1 mice fed malachite green or leucomalachite green for 28 days. (A) Control rat; (B) a rat fed 100 ppm malachite green; (C) a rat fed 600 ppm malachite green; (D) a mouse fed 600 ppm malachite green; and (E) a rat fed 580 ppm leucomalachite green.
week 4. In addition, there were statistically significant linear dose trends for week 3 (P B 0.02) and week 4 (P B0.002). All female mice fed 1160 ppm leucomalachite green had scattered dead or degenerate cells in the transitional epithelium of the urinary bladder (P B 0.001). Many of the cells lacked nuclei, and when visible, the nuclei were condensed or fragmented, suggesting apoptosis. Examination of thin sections revealed that many apparent apoptotic cells were contained within phagocytic vacuoles inside viable epithelial cells. The ISEL technique showed that the cytoplasm of apparently apoptotic cells was moderately positive for the presence of DNA fragments and condensed nuclei stained intensely for DNA fragmentation. Individual cell necrosis was not accompanied by inflammatory changes. Similar apoptosis was not seen in transitional epithelium of the bladders of female mice fed 0, 290, or 580 ppm leucomalachite green.
3.5. DNA adducts and metabolites Male F344 rats and female B6C3F1 mice were fed 0, 100, or 600 ppm malachite green or the similar molar equivalents of leucomalachite green (0, 96, and 580 ppm) for 28 days. At the end of the feeding period, DNA was isolated from the livers and adduct levels were measured using a 32P-postlabeling assay. A single adduct (or
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
163
co-eluting adducts) was observed with both compounds (Fig. 3), with the adduct levels increasing significantly (P B 0.05) as a function of the dose (Fig. 4). With the rats, the hepatic DNA adduct levels did not differ between animals administered malachite green or leucomalachite green. In the mice, however, the highest dose of malachite green (600 ppm) gave a significantly (PB 0.001) higher adduct level than that observed with the nearly equimolar dose (580 ppm) of leucomalachite green. Liver extracts (see Section 2) from the rats were analyzed by HPLC in combination with APCI/MS. Fig. 5 shows total ion and reconstructed ion chromatograms obtained at a low cone voltage (20 V) from a male rat fed 580 ppm leucomalachite green. Under these conditions, the mass spectra consisted primarily of molecular ions (protonated molecules for leucomalachite green and demethylated derivatives, and molecular ions for malachite green N-oxide and demethylated N-oxide derivatives). The reconstructed ion chromatograms show a series of compounds corresponding to leucomalachite green (m/z 331; retention time 13.94 min), its mono-, di-, tri-, and tetra-desmethyl derivatives (m/z 317, 303, 289, and 275; retention times 12.50, 10.71, 8.82, and 6.92), malachite green N-oxide (m/z 345; retention time 9.66 min), and its mono- and di-desmethyl derivatives (m/z 331 and 317; retention times 7.83 and 6.08 min). (A small, but measurable, amount of malachite green was also detected; not shown.) At a higher cone voltage (40 V) collision-induced dissociation gave additional diagnostic fragments. These fragments, which involved losses of dimethylaniline, methyl, or phenyl moieties, are consistent with the fragmentation pathways previously published for leucomalachite green [13]. Additionally, a doserelated increase in leucomalachite green and metabolites was observed in both rat and mouse liver extracts. The analysis of the liver extract from a rat fed 600 ppm malachite green is shown in Fig. 6. The reconstruction ion chromatograms, obtained at a cone voltage of 20 V, show the molecular ions for malachite green (m/z 329; retention time 17.55 min), its mono-, di-, tri-, and tetra-desmethyl derivatives (m/z 315, 301, 287, and 273;
Fig. 4. DNA adduct levels in the livers of F344 rats or B6C3F1 mice fed 0 ppm (open); 100 ppm malachite green or 96 ppm leucomalachite green (hashed); or 600 ppm malachite green or 580 ppm leucomalachite green (solid) for 28 days. Each bar represents the mean 9S.E.M. of 3 – 4 animals. * Significantly (PB 0.05) different from control group.
164
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Fig. 5. LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 580 ppm leucomalachite green (LMG) for 28 days. (A) Mass chromatograms of m/z 345, malachite green N-oxide (MG N-ox); (B) m/z 275, tetradesmethyl LMG; (C) m/z 289, tridesmethyl LMG; (D) m/z 303, didesmethyl LMG; (E) m/z 317; desmethyl LMG (12.5 min) and didesmethyl MG N-ox (6.08 min); (F) m/z 331, LMG (13.94 min) and desmethyl MG N-ox (7.83 min); and (G) total ion chromatogram.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
165
Fig. 6. LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 600 ppm malachite green (MG) for 28 days. (A) Mass chromatograms of m/z 273, tetradesmethyl MG; B) m/z 287, tridesmethyl MG; (C) m/z 301, didesmethyl MG; (D) m/z 315, monodesmethyl MG; (E) m/z 329, MG; and (F) total ion chromatogram.
166
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
retention times 14.78, 12.64, 10.51, and 9.06 min), and malachite green N-oxide (m/z 345; retention time 7.25 min; not shown). A small, but measurable, amount of leucomalachite green was also detected (not shown). As with the leucomalachite green, higher cone voltages produced in-source collision-induced dissociation to give fragments consistent with those previously reported [14]. Likewise, as observed with leucomalachite green extracts, malachite green and metabolites increased with increasing dose. The peak shape for didesmethyl malachite green (retention time 12.64 min; Fig. 6) suggested the presence of two components. The mass spectra for each component at low cone voltage (20 V) showed only ions with m/z 301. At higher cone voltage (60 V), however, the early eluting compound gave a fragment (m/z 194) indicating the loss of methylaniline, whereas the late eluting compound showed losses of dimethylaniline (m/z 180) and aniline (m/z 208) moieties. These fragments are consistent with the presence of both symmetrical and unsymmetrical derivatives of didesmethyl malachite green, with the symmetrical isomer eluting first. This conclusion was confirmed by reacting benzaldehyde with methylaniline and oxidizing with lead oxide, as previously described [13], to give symmetrical didesmethyl malachite green. The mass spectrum of the synthetic material was identical to the early eluting component (not shown). Fig. 1 summarizes the demethylated metabolites observed in the livers of rats fed malachite green or leucomalachite green (the unsymmetrical derivative of didesmethyl malachite green is not shown). Liver extracts were also examined by HPLC in conjunction with UV detection. In rats fed leucomalachite green, the unmetabolized compound was the major product detected in the liver (Fig. 7A), accompanied by small amounts of mono- and di-desmethyl leucomalachite green. Malachite green was the major product detected in the liver of rats and mice fed malachite green (Fig. 7B and C), accompanied by mono- and di-desmethyl malachite green and leucomalachite green, and in the case of rats, mono- and di-desmethyl leucomalachite green.
4. Discussion A comparison of adverse effects suggests that exposure to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. In rats, for example, lower doses of leucomalachite green caused increases in liver to body weight ratios and hepatic vacuolization compared to malachite green. In addition, leucomalachite green caused increases in g-glutamyl transferase, T4, and TSH, effects not observed with malachite green. In mice, leucomalachite green caused significant increases in liver to body weight ratios and urinary bladder apoptosis, effects not observed with malachite green. Histopathological data of the leucomalachite green-fed rats revealed apoptotic follicular epithelial cells in the thyroid gland of two out of eight rats in the 1160 ppm and two out of eight rats in 580 ppm dose groups. Interestingly, follicular cell adenocarcinomas of the thyroid gland were observed in a 2-year feeding study in male and female F344 rats with gentian violet, a triphenylmethane dye similar in
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
167
Fig. 7. Extracts of livers of (A) rats fed 96 (hashed) or 580 (solid) ppm leucomalachite green (LMG); (B) rats fed 100 (hashed) or 600 (solid) ppm malachite green (MG); and (C) mice fed 100 (hashed) or 600 (solid) ppm MG for 28 days. Each bar represents the mean 9S.E.M. from four animals. Des- and didesrefer to the demethylated derivatives of MG or LMG.
structure to malachite green [15]. With leucomalachite green, total T4 levels were decreased, while TSH levels were significantly elevated in male rats fed 1160 ppm leucomalachite green. A decrease in T4 levels is consistent with primary hypothyroidism, but does not preclude other causes of low circulating T4, such as decreased pituitary function, alterations in protein binding of T4, and alterations in peripheral metabolism of T4. However, in combination with an increase in TSH levels, these findings indicate that pituitary function was normal in rats with decreased T4 levels and that primary hypothyroidism was the most likely cause of reduced T4 levels. (In female rats fed 1200 ppm malachite green, decreased T4 levels suggested thyroid dysfunction; however, no decrease in TSH levels was observed.) Previous in vitro studies [13] have shown that leucomalachite green inhibits thyroid peroxidase, the enzyme that catalyzes the iodination and coupling reactions required for thyroid hormone synthesis (Fig. 8). This is consistent with the anti-thyroid effects (de-
168
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
creased T4 and increased TSH) observed in the current study. Therefore, chronic exposure to leucomalachite green could cause thyroid follicular cell tumors through hormonal stimulation by TSH [16]. In leucomalachite green-treated mice, histopathological data revealed that all mice in the 1160 ppm dose group had apoptosis of the transitional epithelium of the urinary bladder. The apoptotic cells were phagocytized by neighboring transitional epithelial cells and appeared to undergo dissolution in phagocytic vacuoles. This apoptotic cell death could be accompanied by a compensatory cell proliferation, which could promote the expansion of initiated cells. 32 P-Postlabeling of liver DNA indicated the formation of a DNA adduct or co-eluting adducts, that increased with increasing dose, in rats and mice fed leucomalachite green or malachite green. In addition, a series of desmethyl derivatives were observed by HPLC/APCI/MS in liver extracts from both species. These results suggest a scenario in which malachite green undergoes a reduction to leucomalachite green or cytochrome P-450 catalyzed N-demethylation to monoand di-desmethyl malachite green (Fig. 8). Leucomalachite green could also undergo a similar N-demethylation by cytochrome P-450. These primary and secondary amine metabolites are similar to carcinogenic arylamines. As such, they
Fig. 8. Proposed mechanism for the metabolism of malachite green (MG) and leucomalachite green (LMG). Brackets indicate hypothesized effects.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
169
could be oxidized to metabolites that react with DNA either directly or after esterification. Misreplication of these lesions may result in mutations that can lead to liver tumors. Furthermore, additional studies [13] have demonstrated the TPOcatalyzed oxidation of leucomalachite green to N-demethylated products, including primary and secondary arylamines (Fig. 8). Preliminary results from our laboratory, using the 32P-postlabeling assay, indicate the presence of a single adduct or co-eluting adducts from thyroid DNA of rats fed leucomalachite green for 28 days (data not shown). This suggests that a genotoxic mechanism for thyroid tumor formation may also be possible. Human exposure to malachite green, through the consumption of treated fish, has been documented in the last few years. Residues up to 35 mg/kg malachite green have been measured in random samples of trout sold in markets in the UK [3]. Perhaps of even greater significance is the conversion of malachite green to and persistence of leucomalachite green in fish tissues. Leucomalachite green residues (9– 96 mg/kg) have been detected in trout marketed for sale at levels 30-fold higher than malachite green residues (0.4–3.4 mg/kg; [14]). In view of the potential adverse effects, as well as the recent trend to include fish as a part of a healthy diet, ascertaining the long term impact and possible activation pathways from exposure to malachite green and leucomalachite green could be beneficial to public health.
Acknowledgements We thank Betty Spadoni and Kathy Carroll for computer support; Stephen Moore for assistance with the animal care services; James Carson, Andrew Matson, Larry Rushing, and Thomas Schmitt for diet preparation and dose analyses; Ralph Patton, Tracy Hagstrom, and Alan Warbritton for assistance with the clinical chemistry; and Ralph Kodell, Charles McCarty, Jeff Gossett, Brett Thorn, Donna Barton, and Jim Parker for assistance with the statistical analyses. This research was supported, in part, by Interagency Agreement c 224-93-0001 between the Food and Drug Administration/National Center for Toxicological Research and the National Institute for Environmental Health Sciences/National Toxicology Program.
References [1] J.G.R. Elferink, H.L. Booij, The action of some triphenylmethane dyes on yeast and erythrocyte membranes, Arzneim.-Forsch. (Drug Res.) 25 (1975) 1248 – 1252. [2] R.A. Schnick, The impetus to register new therapeutants for aquaculture, Prog. Fish-Cult. 50 (1988) 190–196. [3] Veterinary Medicines Directorate, 1995. Annual Report on Surveillance for Veterinary Residues in 1995. Ministry of Agriculture, Fisheries and Food, UK, PB 2756, July 1996. [4] S.J. Culp, F.A. Beland, Malachite green: a toxicological review, J. Am. Coll. Toxicol. 15 (1996) 219–238.
170
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
[5] D.J. Alderman, R.S. Clifton-Hadley, Malachite green: a pharmacokinetic study in rainbow trout, Oncorhynchus mykiss (Walbaum), J. Fish Dis. 16 (1993) 297 – 311. [6] F.C.P. Law, 1994. Total residue depletion and metabolic profile of selected drugs in trout. US Food and Drug Administration Contract c 223-90-7016, Simon Fraser University, Burnaby, BC, Canada V5A 1S6. [7] J.E. Roybal, A.P. Pfenning, R.K. Munns, D.C. Holland, J.A. Hurlbut, A.R. Long, Determination of malachite green and its metabolite, leucomalachite green, in catfish (Ictalurus punctatus) tissue by liquid chromatography with visible detection, J. AOAC Int. 78 (1995) 453 – 457. [8] S.J. Culp, F.A. Beland, Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene, Carcinogenesis 15 (1994) 247 – 252. [9] R.C. Gupta, Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts, Cancer Res. 45 (1985) 5656–5662. [10] J.V. Bradley, Distribution-Free Statistical Tests, Prentice Hall, Englewood Cliffs, NJ, 1968. [11] D.G. Thomas, N. Breslow, J.J. Gart, Trend and homogeneity and analyses of proportions and life table data, Comput. Biomed. Res. 10 (1977) 373 – 381. [12] S.P. Wright, Adjusted P-values for simultaneous inference, Biometrics 48 (1992) 1005 – 1013. [13] D.R. Doerge, H.C. Chang, R.L. Divi, M.I. Churchwell, Mechanism for inhibition of thyroid peroxidase by leucomalachite green, Chem. Res. Toxicol. 11 (1998) 1098 – 1104. [14] D.R. Doerge, M.I. Churchwell, T.A. Gehring, Y.M. Pu, S.M. Plakas, Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry, Rapid Commun. Mass Spectrom. 12 (1998) 1625 – 1634. [15] N.A. Littlefield, 1988. Chronic toxicity and carcinogenicity studies of gentian violet in Fischer 344 rats, NCTR Technical Report for Experiment No. 338. National Center for Toxicological Research, Jefferson, AR 72079. [16] E.D. Williams, TSH and thyroid cancer, Horm. Metab. Res. 23 (1990) 72 – 75.
.
www.elsevier.com/locate/chembiont
Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice Sandra J. Culp a,*, Lonnie R. Blankenship a, Donna F. Kusewitt a,1, Daniel R. Doerge a, Louis T. Mulligan b, Frederick A. Beland a a
National Center for Toxicological Research, HFT-110, 3900 NCTR Road, Jefferson, AR 72079, USA b Center for Veterinary Medicine, FDA, Rock6ille, MD 20855, USA Received 24 March 1999; received in revised form 30 May 1999; accepted 3 June 1999
Abstract 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 metabo-
* Corresponding author. Tel.: + 1-870-543-7941; fax: + 1-870-543-7136. E-mail address: [email protected] (S.J. Culp) 1 Present address: University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. 0009-2797/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 1 9 - 2
154
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
lized 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. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Malachite green; Leucomalachite green; DNA adducts;
32
P-postlabeling; Apoptosis
1. Introduction Malachite green is an N-methylated diaminotriphenylmethane dye (Fig. 1) widely used in the fish and dye industries. The powerful antimicrobial activity of malachite green has been attributed to inhibition of intracellular enzymes, intercalation into DNA, and/or interaction with cellular membranes [1]. Although not approved for use in aquaculture in the USA, malachite green has been used as an antifungal treatment for fish since the 1930s [2]. Fish sold in the USA have not been routinely tested for contamination by malachite green; however, the compound is inexpensive, easy to obtain, and effective, which creates the potential for significant worker and consumer exposure. Random sampling of fish from markets in the UK indicated the continued use of malachite green in the aquaculture industry [3]. Data relating to the carcinogenicity of malachite green are extremely limited, although there is evidence of tumor promotion in rodent liver and suspicion of carcinogenicity based on structure–activity relationships (reviewed in Ref. [4]). Relatively little information is available concerning the metabolism of malachite green. Alderman and Clifton-Hadley [5] studied the uptake, distribution, and elimination of the dye after exposing trout to a 1.6 ppm bath treatment for 40 min. The maximum concentrations of malachite green in the serum, liver, and kidney (ranging from 7.8 to 34.0 ppm) occurred immediately after exposure, while a peak
Fig. 1. Structures of malachite green (MG), leucomalachite green (LMG), and demethylated derivatives.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
155
concentration (10.8 ppm) in the muscle was reached after 90–120 min. Other studies have shown that malachite green is reduced to and persists as leucomalachite green (Fig. 1) in the tissues of fish. For example, Law [6] showed a rapid absorption of malachite green in fingerling trout exposed to 2 ppm for 1 h. The malachite green in the whole fish homogenate decreased with time, while leucomalachite green increased up to 24 h after exposure and remained steady for the next 7 days. In this study, the effects of exposure to malachite green and leucomalachite green have been compared by feeding B6C3F1 mice and Fischer 344 (F344) rats the compounds for 28 days. Our data indicate that leucomalachite green causes a greater number of and more severe changes than malachite green. Examination of DNA and liver extracts indicates that the compounds may be metabolized in a manner similar to carcinogenic aromatic amines.
2. Materials and methods
2.1. Chemicals Malachite green (CAS registry number 569-64-2) and leucomalachite green (CAS registry number 129-73-7) were purchased from Chemsyn, Lenexa, KS. The chemicals were found to be ] 94 and ]98% pure, respectively, by high performance liquid chromatography (HPLC) with UV detection (254 nm) and evaporative light scattering detection, nuclear magnetic resonance spectrometry, atmospheric pressure chemical ionization mass spectrometry (APCI/MS), and elemental analyses. Impurities detected in malachite green were leucomalachite green (1%) and demethlyated derivatives of malachite green (3.5%). The impurities (5 2%) detected in leucomalachite green were monodesmethyl leucomalachite green and malachite green. (9)-Anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide was purchased from Chemsyn.
2.2. Animal treatments Female and male mice (eight per dose group per sex; B6C3F1/Nctr BR (C57BL/ 6N/X/C3H/HeN MTV-) as well as female and male rats (eight per dose group per sex; F344/N Nctr BR) were fed 0, 25, 100, 300, 600, or 1200 ppm malachite green for 28 days. Female mice and male rats (eight per dose group per sex) were fed 0, 290, 580, or 1160 ppm of leucomalachite green for 28 days. The animals were obtained from the breeding colony at the National Center for Toxicological Research, Jefferson, AR, and were 6–7 weeks old at the start of dosing. After the dosing period, blood was collected to measure clinical parameters, and tissues were examined histopathologically. Additional groups of male and female rats were fed 0 or 1200 ppm malachite green for 4 or 21 days (eight per dose group per sex per time point) and blood was collected for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) analyses. Male rats were treated likewise with
156
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
0 or 1160 ppm leucomalachite green (eight per dose group per time point). Another set of male rats and female mice (eight per dose group) was fed 0, 100, or 600 ppm malachite green or 0, 96, or 580 ppm leucomalachite green for 28 days and the livers were analyzed for metabolites and DNA adducts.
2.3. Necropsy and histopathology After 28 days of feeding, the animals were fasted overnight, but had free access to water. The animals were anesthetized with CO2 to collect blood for clinical chemistry measurements. They were then euthanized by CO2 exposure and a gross necropsy was performed. Tissues were fixed in neutral buffered formalin for 48 h, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin. Selected tissues were stained by ISEL (Apoptag kit, Oncor, Gaithersburg, MD), a technique to demonstrate DNA fragmentation. For thin sections, formalin-fixed tissues were embedded in glycol methacrylate, sectioned at 2 mm, and stained with toluidine blue.
2.4. Clinical chemistry Hematology measurements were conducted with a COBAS Minos Vet (Roche Diagnostic Systems, Branchburg, NJ) and included leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelet count, segmented neutrophils, lymphocytes, monocytes, eosinophils, and reticulocyte count. Clinical chemistry measurements were conducted with a COBAS Mira Plus (Roche Diagnostic Systems) and included total protein and bile acids, blood urea nitrogen, creatinine, alanine aminotransferase, and alkaline phosphatase. In rats, aspartate amino transferase, glucose, cholesterol, triglycerides, g-glutamyl transferase, albumin, sorbitol dehydrogenase, creatine kinase, sodium, potassium, chloride, calcium, and phosphorus were also measured. Total T3 and total T4 were determined with a ‘Coat-A-Count’ procedure obtained from DPC, Los Angeles, CA. The procedure is a solid-phase radioimmunoassay, wherein 125I-labeled T3/T4 competes with the sample for antibody sites. TSH was measured with a double antibody RIA procedure. Freshly prepared [125I]TSH was allowed to react overnight with the specific antibody and sample. An excess of second antibody containing polyethylene glycol was then added, bound and unbound [125I]hormone were separated by centrifugation, and the radioactivity was measured in the precipitates. The amount of sample was calculated from a standard curve.
2.5. Li6er extraction and analyses For HPLC analyses the livers were extracted using a method modified from Roybal et al. [7]. Specifically, 1 –10 g of liver was homogenized in a glass-Teflon homogenizer in a mixture of 2 ml 250 mg/ml hydroxylamine HCl in water, 3 ml 50
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
157
mM toluene sulfonic acid, 10 ml 100 mM ammonium acetate (pH 4.5), and 30 ml acetonitrile. Sodium chloride (2 g) was added and the samples were centrifuged for 10 min at 3000 rpm at 25°C. The supernatant was extracted with a mixture of 40 ml water, 2 ml diethylene glycol, and 90 ml methylene chloride. The organic layer was then concentrated to approximately 1 ml. Solid phase extraction was conducted as described in Roybal et al. [7]. The samples were analyzed by HPLC using a CN guard column, a 4.6 ×250 mm Spherisorb CN column, and a 20× 2.0 mm i.d. PbO2 oxidative post-column coupled to a Waters model 996 photodiode array detector. The following elution conditions were used with a flow rate of 1 ml/min: 0–10 min, 60% A, 40% B; 10 – 25 min a linear gradient to 50% A, 50% B (solvent A, 100 mM ammonium acetate (pH 4.5); solvent B, acetonitrile), and monitoring was conducted at 618 nm. The samples were quantified by comparison to malachite green or leucomalachite green standards. Untreated rat livers were spiked with the standards and processed in the same manner as treated livers to generate a calibration curve. No interconversion of the standards was observed in the spiked samples. The samples were also analyzed by HPLC in combination with APCI/MS. The separations were performed with a Dionex GP40 pump (Dionex, Sunnyvale, CA) and either a manual Rheodyne 7125 injector (Rheodyne, Cotati, CA) or a Dionex AS3500 autosampler. A Prodigy ODS-3 column (5 mm, 4.6× 250 mm) was used for separation of leucomalachite green and metabolites. The flow rate was 1.0 ml/min and a 10-min linear gradient was run from 50% acetonitrile in 50 mM ammonium acetate (pH 4.5), to 100% acetonitrile. Separation of malachite green and metabolites was performed using a Spherisorb S5 Nitrile column (80A, 5 mm, 4.6 × 250 mm) and a solvent system containing 40% acetonitrile in 50 mM ammonium acetate (pH 4.5), at a flow rate of 1.0 ml/min. For mass spectrometry, a VG Platform II single quadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with an APCI interface was used. The total HPLC column effluent was delivered into the ion source (150°C) through a heated nebulizer probe (500°C) using nitrogen as the probe and bath gas. Positive ions were acquired in full scan mode (m/z 100–800 in 1.5 s cycle time) and a UV detector set at 260 nm was placed in-line before the mass spectrometer. Two separate scan functions with different sampling cone-skimmer potentials were used to acquire mass spectra with minimal (20 V) or significant (60 V) amounts of fragmentation through in-source collision-induced dissociation. The desmethyl derivatives were synthesized (unpublished data) to confirm their presence in the samples subjected to APCI/MS. The samples were quantified by comparison to the response of the malachite and leucomalachite green standards.
2.6. DNA isolation and
32
P-postlabeling
DNA was isolated from the liver by the method reported in Culp and Beland [8]. Approximately 10 mg of DNA was 32P-postlabeled using n-butanol enrichment [9]. Adducts were separated by thin layer chromatography performed on 0.1 mm Machery Nagel 300 polyethylene imine cellulose plates (Alltech, Deerfield, IL) using the following solvent directions, D1: 0.9 M sodium phosphate (pH 6.8); D2: 3.6 M
158
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
lithium formate, 8.5 M urea (pH 3.5); D3: 1.2 M lithium chloride, 0.5 M Tris–HCl, 8 M urea (pH 8.0). A final wash was conducted in D3 with the solvent used in D1. DNA adducts were visualized using a Storm 860 phosphor imaging system (Molecular Dynamics, Sunnyvale, CA). The adduct levels were quantified by comparison to a 10b-(deoxyguanosin-N 2-yl)-7b,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene standard, obtained by reacting DNA with (9 )-anti-benzo[a]pyrene-trans7,8-dihydrodiol-9,10-epoxide.
2.7. Data analysis Body weights were measured weekly for a total of five measurements per animal and were analyzed on a per cage basis. A mixed-models approach to repeated measures analysis was used to model the mean cage body weights. The weights were modeled using the fixed effects of dose, time, and dose-by-time interaction. Dunnett’s two-sided test was used to test for differences between the control group means and each treatment group means. Contrasts were used to test for linear dose trends. A SAS GLM procedure was used to model the organ weights and the ratios of organ weights to body weights as functions of the dose. Dunnett’s test was used to test for differences between the control group mean and each treatment group mean. Clinical and hematology data were analyzed using a one-way analysis of variance (ANOVA) and Dunnett’s test. In instances where there was a non-normal distribution and/or an unequal variance, the analyses were conducted using a Kruskal– Wallis one-way ANOVA on ranks, with differences between the control group median and each treatment group median being tested by Dunnett’s method. DNA adduct levels were analyzed by one-way ANOVA, Dunnett’s test, and regression analysis. When an unequal variance occurred, the data were transformed by obtaining the square root before conducting the analysis Fisher’s exact test [10] was used to compare the proportion of lesions in the control group to that in each of the dosed groups. The Cochran–Armitage test [11] for a dose-response trend on proportions was used when all dose groups were examined. An exact test for this procedure was used because there were less than 10 animals in each dose group. All tests are one-sided in that decreases in incidence with an increasing dose were not considered. The P-values were adjusted with a modified Bonferroni procedure designed by Holm and modified by Wright [12]. 3. Results
3.1. Rats fed malachite green In female rats there were significant decreases in the mean body weights in the 1200 ppm dose group for weeks 1–4, with the animals weighing 80–83% of the control rats. Although the male rats fed 1200 ppm malachite green tended to have lower body weights (82 – 87%), as compared to the control group, the differences were not significant.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
159
Fig. 2. Liver to body weight ratios in (A) female and (B) male F344 rats fed malachite green or (C) male rats fed leucomalachite green for 28 days. Each data point is the mean 9 S.E.M. of eight animals. * Significantly (P B0.05) different from control group.
In the female rats, the animals in the three highest doses (300, 600, and 1200 ppm) of malachite green had significantly increased ratios of liver weights to body weights (Fig. 2A). The ratio of liver weight to body weight was significantly increased in the male rats fed 600 and 1200 ppm malachite green as compared to the control group (Fig. 2B). In both sexes, there was a significant linear increasing trend in the levels of g-glutamyl transferase, with the value in the 1200 ppm female dose group being 4.2-fold greater (P B0.0005) than that in the controls. Blood hematology measurements in female rats showed slight ( B7%), but significant, decreases in the 1200 ppm dose group in erythrocyte count, hemoglobin, hematocrit, mean erythrocyte hemoglobin, and mean erythrocyte concentration. Male rats had slight (B 3%), but significant, decreases in mean erythrocyte hemoglobin in the 300, 600, and 1200 ppm dose groups. Additional groups of female and male rats were fed 0 or 1200 ppm malachite green for 4 or 21 days and blood was collected for T3, T4, and TSH measurements. The T3 levels were significantly higher in female rats fed 1200 ppm malachite green as compared to the control group on day 21 (Table 1). The T4 levels were significantly lower on both days 4 and 21 in the female rats in the 1200 ppm group as compared to the respective control groups. There were no significant changes in T3 or T4 levels in males or in the TSH levels in either sex.
160
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Seven out of eight female rats fed 1200 ppm malachite green had minimal to mild hepatocyte vacuolization (P B0.01). The same lesion, primarily midzonal in location, was observed in one of eight male rats fed 600 ppm malachite green and four of eight male rats fed the 1200 ppm dose.
3.2. Male rats fed leucomalachite green Male rats fed 1160 ppm leucomalachite green had significantly lower body weights (91 – 92% of the control group) at weeks 2, 3, and 4. There were also Table 1 T3, T4, and TSH levels in female and male F344 rats fed malachite green or leucomalachite green for 4 or 21 daysa Clinical parameter
Compound
T3 (ng/dl)
Malachite green Leucomalachite green Malachite green
Time (days)
4
21
Leucomalachite green T4 (mg/dl)
Malachite green
4
21
Leucomalachite green TSH (ng/ml)
Malachite green
4
Leucomalachite green Malachite green Leucomalachite green
Dose (ppm) (mean 9S.E.M.)
Female Male
0 116.2 95.41 128.3 96.53
1200/1160 118.7 9 6.13 134.7 96.09
Male
113.7 9 5.25
108.0 94.35
Female Male
105.4 9 2.21 106.0 9 5.21
123.1 9 4.12b 111.1 97.60
97.5 9 3.56
98.5 94.06
Male
Leucomalachite green Malachite green
Sex
Female Male
3.1 9 0.14 3.8 9 0.17
2.6 90.14b 3.7 90.05
Male
5.0 9 0.29
3.4 90.18b
Female Male
3.0 9 0.14 3.5 9 0.05
2.5 90.11b 3.1 90.04
Male
3.0 9 0.17
2.3 90.13b
0.86 9 0.12 0.73 9 0.06
1.0 90.12 1.1 9 0.17
1.9 90.16
3.0 9 0.44b
Female Male Male
21
Female Male Male
0.75 9 0.09 1.2 90.15 3.7 90.80
0.97 9 0.13 1.2 9 0.17 6.3 9 1.58b
a Female and male F344 rats were fed 0 or 1200 ppm malachite green and male rats were fed 0 or 1160 ppm leucomalachite green for 4 or 21 days (eight per dose group per sex per time point). Blood was collected for T3, T4, and TSH analyses. The data are expressed as the mean9S.E.M. of 7–8 rats. b Significantly (PB0.05) different from control group at the same time point.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
161
significant decreases in body weights in the 580 ppm dose group at weeks 3 and 4 (94% of the control group). Linear decreasing dose trends were observed for weeks 2, 3, and 4 (P B0.003). The ratio of liver weights to body weights was significantly increased for all three dose groups as compared to the control group (Fig. 2C). g-Glutamyl transferase levels were 2.2-fold higher (PB 0.05) and phosphorus levels were slightly increased (10%; PB 0.05) in male rats fed 1160 ppm leucomalachite green. In addition, erythrocyte count, hemoglobin, and hematocrit levels showed slight (B6%), but significant, decreases from the controls in the 1160 ppm dose group. An additional group of male rats was fed 0 or 1160 ppm leucomalachite green for 4 or 21 days and blood was collected for T3, T4, and TSH measurements. There was a significant decrease in T4 and increase in TSH levels on days 4 and 21 as compared to the respective control groups (Table 1). In male rats fed leucomalachite green, hepatocyte vacuolization, primarily midzonal and centrilobular in location, was seen in 7/8 rats fed 1160 ppm (P B 0.005), 5/8 rats fed 580 ppm (P B0.04), and 2/8 rats fed 290 ppm, a significant dose trend (P B 0.0004). Two of the eight rats fed 1160 ppm and two of the eight rats fed 580 ppm leucomalachite green had apoptotic follicular epithelial cells in the thyroid gland. Morphologic changes consisted of sloughed follicular cells with condensed nuclei located within the follicles. An inflammatory reaction was not present. There was evidence of follicular epithelium regeneration, since even the most severely affected follicles were still lined by viable epithelium.
3.3. Mice fed malachite green Female mice fed 1200 ppm malachite green had significantly lower body weights (91– 92% of control group) at weeks 3 (PB 0.02) and 4 (PB 0.03). The body weights of male mice were not significantly effected by any of the dose levels of malachite green. In female mice fed 600 or 1200 ppm malachite green, there were slight (6–8%) significant decreases in the erythrocyte count and hemoglobin and hematocrit levels as compared to the control group; in male mice, significant decreases in these parameters were observed in the 1200 ppm dose group. The mean erythrocyte volume was increased 1 – 2% (PB 0.05) in female mice fed 300, 600, and 1200 ppm malachite green as compared to the control group. There was also a 1.4–1.9-fold increase in reticulocytes in these groups (PB 0.05). Male mice fed 1200 ppm malachite green showed a 1.6-fold increase in reticulocytes (P B 0.05). There were no significant histopathological changes observed in the mice fed malachite green.
3.4. Female mice fed leucomalachite green Female mice fed 1160 ppm leucomalachite green had significantly lower body weights (93% of the control group) at week 4. A marginally significant decrease (PB 0.01) from the control group was observed in the female mice fed 580 ppm at
162
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Fig. 3. 32P-Postlabeling scans of liver DNA from F344 rats or B6C3F1 mice fed malachite green or leucomalachite green for 28 days. (A) Control rat; (B) a rat fed 100 ppm malachite green; (C) a rat fed 600 ppm malachite green; (D) a mouse fed 600 ppm malachite green; and (E) a rat fed 580 ppm leucomalachite green.
week 4. In addition, there were statistically significant linear dose trends for week 3 (P B 0.02) and week 4 (P B0.002). All female mice fed 1160 ppm leucomalachite green had scattered dead or degenerate cells in the transitional epithelium of the urinary bladder (P B 0.001). Many of the cells lacked nuclei, and when visible, the nuclei were condensed or fragmented, suggesting apoptosis. Examination of thin sections revealed that many apparent apoptotic cells were contained within phagocytic vacuoles inside viable epithelial cells. The ISEL technique showed that the cytoplasm of apparently apoptotic cells was moderately positive for the presence of DNA fragments and condensed nuclei stained intensely for DNA fragmentation. Individual cell necrosis was not accompanied by inflammatory changes. Similar apoptosis was not seen in transitional epithelium of the bladders of female mice fed 0, 290, or 580 ppm leucomalachite green.
3.5. DNA adducts and metabolites Male F344 rats and female B6C3F1 mice were fed 0, 100, or 600 ppm malachite green or the similar molar equivalents of leucomalachite green (0, 96, and 580 ppm) for 28 days. At the end of the feeding period, DNA was isolated from the livers and adduct levels were measured using a 32P-postlabeling assay. A single adduct (or
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
163
co-eluting adducts) was observed with both compounds (Fig. 3), with the adduct levels increasing significantly (P B 0.05) as a function of the dose (Fig. 4). With the rats, the hepatic DNA adduct levels did not differ between animals administered malachite green or leucomalachite green. In the mice, however, the highest dose of malachite green (600 ppm) gave a significantly (PB 0.001) higher adduct level than that observed with the nearly equimolar dose (580 ppm) of leucomalachite green. Liver extracts (see Section 2) from the rats were analyzed by HPLC in combination with APCI/MS. Fig. 5 shows total ion and reconstructed ion chromatograms obtained at a low cone voltage (20 V) from a male rat fed 580 ppm leucomalachite green. Under these conditions, the mass spectra consisted primarily of molecular ions (protonated molecules for leucomalachite green and demethylated derivatives, and molecular ions for malachite green N-oxide and demethylated N-oxide derivatives). The reconstructed ion chromatograms show a series of compounds corresponding to leucomalachite green (m/z 331; retention time 13.94 min), its mono-, di-, tri-, and tetra-desmethyl derivatives (m/z 317, 303, 289, and 275; retention times 12.50, 10.71, 8.82, and 6.92), malachite green N-oxide (m/z 345; retention time 9.66 min), and its mono- and di-desmethyl derivatives (m/z 331 and 317; retention times 7.83 and 6.08 min). (A small, but measurable, amount of malachite green was also detected; not shown.) At a higher cone voltage (40 V) collision-induced dissociation gave additional diagnostic fragments. These fragments, which involved losses of dimethylaniline, methyl, or phenyl moieties, are consistent with the fragmentation pathways previously published for leucomalachite green [13]. Additionally, a doserelated increase in leucomalachite green and metabolites was observed in both rat and mouse liver extracts. The analysis of the liver extract from a rat fed 600 ppm malachite green is shown in Fig. 6. The reconstruction ion chromatograms, obtained at a cone voltage of 20 V, show the molecular ions for malachite green (m/z 329; retention time 17.55 min), its mono-, di-, tri-, and tetra-desmethyl derivatives (m/z 315, 301, 287, and 273;
Fig. 4. DNA adduct levels in the livers of F344 rats or B6C3F1 mice fed 0 ppm (open); 100 ppm malachite green or 96 ppm leucomalachite green (hashed); or 600 ppm malachite green or 580 ppm leucomalachite green (solid) for 28 days. Each bar represents the mean 9S.E.M. of 3 – 4 animals. * Significantly (PB 0.05) different from control group.
164
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
Fig. 5. LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 580 ppm leucomalachite green (LMG) for 28 days. (A) Mass chromatograms of m/z 345, malachite green N-oxide (MG N-ox); (B) m/z 275, tetradesmethyl LMG; (C) m/z 289, tridesmethyl LMG; (D) m/z 303, didesmethyl LMG; (E) m/z 317; desmethyl LMG (12.5 min) and didesmethyl MG N-ox (6.08 min); (F) m/z 331, LMG (13.94 min) and desmethyl MG N-ox (7.83 min); and (G) total ion chromatogram.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
165
Fig. 6. LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 600 ppm malachite green (MG) for 28 days. (A) Mass chromatograms of m/z 273, tetradesmethyl MG; B) m/z 287, tridesmethyl MG; (C) m/z 301, didesmethyl MG; (D) m/z 315, monodesmethyl MG; (E) m/z 329, MG; and (F) total ion chromatogram.
166
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
retention times 14.78, 12.64, 10.51, and 9.06 min), and malachite green N-oxide (m/z 345; retention time 7.25 min; not shown). A small, but measurable, amount of leucomalachite green was also detected (not shown). As with the leucomalachite green, higher cone voltages produced in-source collision-induced dissociation to give fragments consistent with those previously reported [14]. Likewise, as observed with leucomalachite green extracts, malachite green and metabolites increased with increasing dose. The peak shape for didesmethyl malachite green (retention time 12.64 min; Fig. 6) suggested the presence of two components. The mass spectra for each component at low cone voltage (20 V) showed only ions with m/z 301. At higher cone voltage (60 V), however, the early eluting compound gave a fragment (m/z 194) indicating the loss of methylaniline, whereas the late eluting compound showed losses of dimethylaniline (m/z 180) and aniline (m/z 208) moieties. These fragments are consistent with the presence of both symmetrical and unsymmetrical derivatives of didesmethyl malachite green, with the symmetrical isomer eluting first. This conclusion was confirmed by reacting benzaldehyde with methylaniline and oxidizing with lead oxide, as previously described [13], to give symmetrical didesmethyl malachite green. The mass spectrum of the synthetic material was identical to the early eluting component (not shown). Fig. 1 summarizes the demethylated metabolites observed in the livers of rats fed malachite green or leucomalachite green (the unsymmetrical derivative of didesmethyl malachite green is not shown). Liver extracts were also examined by HPLC in conjunction with UV detection. In rats fed leucomalachite green, the unmetabolized compound was the major product detected in the liver (Fig. 7A), accompanied by small amounts of mono- and di-desmethyl leucomalachite green. Malachite green was the major product detected in the liver of rats and mice fed malachite green (Fig. 7B and C), accompanied by mono- and di-desmethyl malachite green and leucomalachite green, and in the case of rats, mono- and di-desmethyl leucomalachite green.
4. Discussion A comparison of adverse effects suggests that exposure to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. In rats, for example, lower doses of leucomalachite green caused increases in liver to body weight ratios and hepatic vacuolization compared to malachite green. In addition, leucomalachite green caused increases in g-glutamyl transferase, T4, and TSH, effects not observed with malachite green. In mice, leucomalachite green caused significant increases in liver to body weight ratios and urinary bladder apoptosis, effects not observed with malachite green. Histopathological data of the leucomalachite green-fed rats revealed apoptotic follicular epithelial cells in the thyroid gland of two out of eight rats in the 1160 ppm and two out of eight rats in 580 ppm dose groups. Interestingly, follicular cell adenocarcinomas of the thyroid gland were observed in a 2-year feeding study in male and female F344 rats with gentian violet, a triphenylmethane dye similar in
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
167
Fig. 7. Extracts of livers of (A) rats fed 96 (hashed) or 580 (solid) ppm leucomalachite green (LMG); (B) rats fed 100 (hashed) or 600 (solid) ppm malachite green (MG); and (C) mice fed 100 (hashed) or 600 (solid) ppm MG for 28 days. Each bar represents the mean 9S.E.M. from four animals. Des- and didesrefer to the demethylated derivatives of MG or LMG.
structure to malachite green [15]. With leucomalachite green, total T4 levels were decreased, while TSH levels were significantly elevated in male rats fed 1160 ppm leucomalachite green. A decrease in T4 levels is consistent with primary hypothyroidism, but does not preclude other causes of low circulating T4, such as decreased pituitary function, alterations in protein binding of T4, and alterations in peripheral metabolism of T4. However, in combination with an increase in TSH levels, these findings indicate that pituitary function was normal in rats with decreased T4 levels and that primary hypothyroidism was the most likely cause of reduced T4 levels. (In female rats fed 1200 ppm malachite green, decreased T4 levels suggested thyroid dysfunction; however, no decrease in TSH levels was observed.) Previous in vitro studies [13] have shown that leucomalachite green inhibits thyroid peroxidase, the enzyme that catalyzes the iodination and coupling reactions required for thyroid hormone synthesis (Fig. 8). This is consistent with the anti-thyroid effects (de-
168
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
creased T4 and increased TSH) observed in the current study. Therefore, chronic exposure to leucomalachite green could cause thyroid follicular cell tumors through hormonal stimulation by TSH [16]. In leucomalachite green-treated mice, histopathological data revealed that all mice in the 1160 ppm dose group had apoptosis of the transitional epithelium of the urinary bladder. The apoptotic cells were phagocytized by neighboring transitional epithelial cells and appeared to undergo dissolution in phagocytic vacuoles. This apoptotic cell death could be accompanied by a compensatory cell proliferation, which could promote the expansion of initiated cells. 32 P-Postlabeling of liver DNA indicated the formation of a DNA adduct or co-eluting adducts, that increased with increasing dose, in rats and mice fed leucomalachite green or malachite green. In addition, a series of desmethyl derivatives were observed by HPLC/APCI/MS in liver extracts from both species. These results suggest a scenario in which malachite green undergoes a reduction to leucomalachite green or cytochrome P-450 catalyzed N-demethylation to monoand di-desmethyl malachite green (Fig. 8). Leucomalachite green could also undergo a similar N-demethylation by cytochrome P-450. These primary and secondary amine metabolites are similar to carcinogenic arylamines. As such, they
Fig. 8. Proposed mechanism for the metabolism of malachite green (MG) and leucomalachite green (LMG). Brackets indicate hypothesized effects.
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
169
could be oxidized to metabolites that react with DNA either directly or after esterification. Misreplication of these lesions may result in mutations that can lead to liver tumors. Furthermore, additional studies [13] have demonstrated the TPOcatalyzed oxidation of leucomalachite green to N-demethylated products, including primary and secondary arylamines (Fig. 8). Preliminary results from our laboratory, using the 32P-postlabeling assay, indicate the presence of a single adduct or co-eluting adducts from thyroid DNA of rats fed leucomalachite green for 28 days (data not shown). This suggests that a genotoxic mechanism for thyroid tumor formation may also be possible. Human exposure to malachite green, through the consumption of treated fish, has been documented in the last few years. Residues up to 35 mg/kg malachite green have been measured in random samples of trout sold in markets in the UK [3]. Perhaps of even greater significance is the conversion of malachite green to and persistence of leucomalachite green in fish tissues. Leucomalachite green residues (9– 96 mg/kg) have been detected in trout marketed for sale at levels 30-fold higher than malachite green residues (0.4–3.4 mg/kg; [14]). In view of the potential adverse effects, as well as the recent trend to include fish as a part of a healthy diet, ascertaining the long term impact and possible activation pathways from exposure to malachite green and leucomalachite green could be beneficial to public health.
Acknowledgements We thank Betty Spadoni and Kathy Carroll for computer support; Stephen Moore for assistance with the animal care services; James Carson, Andrew Matson, Larry Rushing, and Thomas Schmitt for diet preparation and dose analyses; Ralph Patton, Tracy Hagstrom, and Alan Warbritton for assistance with the clinical chemistry; and Ralph Kodell, Charles McCarty, Jeff Gossett, Brett Thorn, Donna Barton, and Jim Parker for assistance with the statistical analyses. This research was supported, in part, by Interagency Agreement c 224-93-0001 between the Food and Drug Administration/National Center for Toxicological Research and the National Institute for Environmental Health Sciences/National Toxicology Program.
References [1] J.G.R. Elferink, H.L. Booij, The action of some triphenylmethane dyes on yeast and erythrocyte membranes, Arzneim.-Forsch. (Drug Res.) 25 (1975) 1248 – 1252. [2] R.A. Schnick, The impetus to register new therapeutants for aquaculture, Prog. Fish-Cult. 50 (1988) 190–196. [3] Veterinary Medicines Directorate, 1995. Annual Report on Surveillance for Veterinary Residues in 1995. Ministry of Agriculture, Fisheries and Food, UK, PB 2756, July 1996. [4] S.J. Culp, F.A. Beland, Malachite green: a toxicological review, J. Am. Coll. Toxicol. 15 (1996) 219–238.
170
S.J. Culp et al. / Chemico-Biological Interactions 122 (1999) 153–170
[5] D.J. Alderman, R.S. Clifton-Hadley, Malachite green: a pharmacokinetic study in rainbow trout, Oncorhynchus mykiss (Walbaum), J. Fish Dis. 16 (1993) 297 – 311. [6] F.C.P. Law, 1994. Total residue depletion and metabolic profile of selected drugs in trout. US Food and Drug Administration Contract c 223-90-7016, Simon Fraser University, Burnaby, BC, Canada V5A 1S6. [7] J.E. Roybal, A.P. Pfenning, R.K. Munns, D.C. Holland, J.A. Hurlbut, A.R. Long, Determination of malachite green and its metabolite, leucomalachite green, in catfish (Ictalurus punctatus) tissue by liquid chromatography with visible detection, J. AOAC Int. 78 (1995) 453 – 457. [8] S.J. Culp, F.A. Beland, Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene, Carcinogenesis 15 (1994) 247 – 252. [9] R.C. Gupta, Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts, Cancer Res. 45 (1985) 5656–5662. [10] J.V. Bradley, Distribution-Free Statistical Tests, Prentice Hall, Englewood Cliffs, NJ, 1968. [11] D.G. Thomas, N. Breslow, J.J. Gart, Trend and homogeneity and analyses of proportions and life table data, Comput. Biomed. Res. 10 (1977) 373 – 381. [12] S.P. Wright, Adjusted P-values for simultaneous inference, Biometrics 48 (1992) 1005 – 1013. [13] D.R. Doerge, H.C. Chang, R.L. Divi, M.I. Churchwell, Mechanism for inhibition of thyroid peroxidase by leucomalachite green, Chem. Res. Toxicol. 11 (1998) 1098 – 1104. [14] D.R. Doerge, M.I. Churchwell, T.A. Gehring, Y.M. Pu, S.M. Plakas, Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry, Rapid Commun. Mass Spectrom. 12 (1998) 1625 – 1634. [15] N.A. Littlefield, 1988. Chronic toxicity and carcinogenicity studies of gentian violet in Fischer 344 rats, NCTR Technical Report for Experiment No. 338. National Center for Toxicological Research, Jefferson, AR 72079. [16] E.D. Williams, TSH and thyroid cancer, Horm. Metab. Res. 23 (1990) 72 – 75.
.