Effect of Dietary Fat on Selected Parameters of Toxicity Following 1- or 3-Month Exposure of Rats to Toluidine Isomers

Abstract

The aim of the study was to investigate the effect of the dietary fat on selected parameters of toluidines toxicity in rats during sub-chronic exposure. Three isomers of toluidine (ortho, meta, and para) were administered to rats in the diet for 1 and 3 months at levels 40, 80, 160 mg/kg/day in two kinds of diet containing either 4% or 14% fat. All doses of toluidine isomers produced a 1.5- to 9.8-fold increase in methemoglobin (MetHb) level during both treatment periods. A distinct dose-response relationship was observed, especially for o- and m-toluidine; the effect was generally greater in rats fed high-fat diet. Reduced glutathione level in liver was increased in all treated groups, 1.5- to 5.1-fold, irrespective of the kind of diet. An increase in hepatic lipid peroxidation (thiobarbituric acid reactive substances; TBARS), 1.5- to 4.5-fold, was noticed in the majority of the treated groups. Generally, there was no consistent effect of diet except for p-toluidine where the level of hepatic TBARS was lower in rats fed high-fat diet. Blood urea nitrogen (BUN) level in animals treated with all doses of o- and m-toluidine was 1.3- to 5.0-fold higher in comparison with respective controls. No clear relationship between BUN level and the kind of diet was found. No effect of toluidines on the activity of serum aspartate aminotrans-ferase (AST) and sorbitol dehydrogenase (SDH) were observed. In the majority of groups treated for 30 and 90 days the amount of toluidines in 24-h urine was lower in rats fed high-fat diet. Final body weight gain in rats treated with o- and p-toluidine (80 and 160 mg/kg body weight [b.w.]) was lower as compared to controls. In summary the high-fat diet stimulated methemoglobin formation in rats treated with o- and m-toluidine and cause the decrease in the amount of toluidines in 24-h urine. The high content of fat did not affect consistently the other parameters tested.

Diet and nutrition are well known to have profound effects on the pharmacological and toxicological responses of laboratory animals to drugs and environmental chemicals (Parke and Ioannides 1994). Among components of diet, lipids play a very significant role and have received great attention. Lipids are necessary for the synthesis and functioning of biological membranes and for the biosynthesis of prostaglandins and other prostanoids. On the other hand, increased dietary intake of polyunsaturated fatty acids (PUFAs) stimulates lipid peroxidation and activation of carcinogens (Kwei and Bjeldanes 1990). Dietary lipids affect xenobiotic biotransformation by modulation the constitutive and inducible levels of specific cytochrome P450 isozymes (Ioannides 1999). High-fat diet can also increase the activity of phase II enzymes, which are essential for many xenobiotics detoxication e.g., epoxide hydrolase, UDP-glucuronosyl transferase and glutathione S-transferase (Yang et al. 1993). The basis for dietary-induced changes in the activity of xenobiotic metabolizing enzymes is not definitely explained. It is known that dietary lipids regulate levels of fatty acids in cells. Diet-induced changes in cellular fatty acids may be responsible for differences in levels of xenobiotic metabolizing enzymes (Yang et al. 1993). There is also evidence that fatty acids regulate the expression of selected genes, although the precise mechanism for this effect is undefined (Mc Donough, Stukey, and Martin 1992).

Toluidine isomers have been of commercial use for over a century, in the manufacturing of dyes, pigments, rubber, pesticides, and textile auxiliares and in pharmaceutical industry (Brock, Hundley, and Lieder 1990). Very few data concerning toxicity of toluidines can be found in available literature. The rat oral LD₅₀ values are as follows: 670 mg/kg for o-toluidine, 450 mg/kg for m-toluidine (Lewis 1996), and 656 mg/kg for p-toluidine (Clayton and Clayton 1994). Almost all information refers to o-toluidine, which has attracted great interest because of its carcinogenicity (IARC 2000). In contrast to o-toluidine, no significant carcinogenic or genotoxic activity was shown for the meta and para isomers (Weisburger et al. 1978). Administration of toluidine isomers to rats caused paraparesis, convulsions, drowsiness, and hematuria. Methemoglobin formation was also noted, the meta isomer was found to be the most potent (Seńczuk and Rucińska 1984a). Toxic effects of o-toluidine include reticulocytosis and anemia in rats and mice (Lunkin 1967) as well as formation of keratosis and metaplasia in the epithelium in the bladder of rats (Ekman and Strömbeck 1947).

Biotransformation of toluidine isomers is very similar and proceeds primarily through ring hydroxylation with subsequent conjugation. The main urine matabolites are aminomethylphenols (Cheever et al. 1980; Son, Everett, and Fiala 1980).

The aim of the study was to examine whether the content of fat in the diet can affect selected biochemical parameters in rats during subchronic exposure to toluidine isomers.

MATERIALS AND METHODS

Experimental Design

Male Wistar rats, 220 ± 10 g, bred in the animal facility of the Department of Toxicology, University of Medical Sciences in Poznań, were used in the experiment. Rats were housed in plastic cages, four rats per cage, in a room maintained at 22°C ± 2°C, a relative humidity 50% to 60%, and 12-h light-dark cycle. Feed and water were available ad libitum. Three isomers of toluidine were administered in a diet for 1 and 3 months at levels approximately 40, 80, 160 mg/kg body weight [b.w.] day. o-Toluidine (99.5%) and m-toluidine (99%) were obtained from Merck, p-toluidine (99.7%) from Sigma-Aldrich. We selected the same doses as were used in the previous study reported by Malik-Bryś and Seńczuk (1995). The doses were high enough to affect some biochemical parameters but did not cause the death of rats during chronic treatment. Each isomer was administered in two kinds of diet: standard diet (certified ISO 9001 laboratory feed “Labofeed H” from Feeds and Concentrates Production Plant, Kcynia, Poland) containing 4% fat and high-fat diet containing additionally 10% sunflower oil (commercially available). Each group consisted of eight rats. Control rats received the same two kinds of diet without toluidines. The diet was given to rats in the form of pulp globules after being worked up with water. Toluidine solutions were added to the diet at concentrations calculated to provide the intended intakes in mg/kg/day. Content of toluidines in diet and homogenicity were determined every two weeks. (Jodynis-Liebert and Bennasir 2000). The average concentration of toluidine isomers in diet during the experiment is shown in Table 1. Throughout the study the general health of animals was checked. The animals were weighed, the food and water intake was recorded every 3 day. On the basis of these data the amounts of toluidines added to the diet were calculated. The calculated consumed doses of toluidines are presented in Table 1. During the experiment rats were placed into metabolic cages every week (1-month treatment) or every 2 weeks (3-month treatment) and 24-h urine was collected for the determination of toluidines. At the end of the treatment rats were killed by exsanguination via cardiac puncture under narcotan anaesthesia. Plasma was separated and liver was perfused with cold 1.15% KCl. Liver samples were stored at –70°C until analyzed. The following liver tissue homogenates were prepared: in 3 volumes of phosphate buffer, pH 8, for glutathione assay, and in 19 volumes of 1.15% KCl-phosphate buffer pH 7.4 (1:1) for lipid peroxidation assay.

The experiment was performed according to the Local Animal Ethics Committee guidelines for animal experimentation.

Biochemical Assays

Methemoglobin (MetHb) level was determined by Evelyn-Malloy method (Mc Lean et al. 1967). In one part of blood sample, the absorbance of the sum of methemoglobin and hemoglobin converted to methemoglobin by potassium ferricyanide was measured spectrophotometrically, in the other part, only absorbance of present methemoglobin. The measurement was repeated after addition of sodium cyanide to both samples which caused cyanomethemoglobin formation and disappearance of MetHb absorbance. Hepatic glutathione (GSH) was determined by the method described by Sedlak and Lindsay (1968) with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic) acid). A marker of lipid peroxidation, malondialdehyde level, was measured in the liver by the thiobarbituric acid reactive substances (TBARS) assay (Hu, Frankel, and Tappel 1990). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH) activities, and blood urea nitrogen (BUN) level were measured using analytical kits (Pointe Scientific, Poland; Sigma Diagnostics). Aminotransferase activity was determined by the routine Reitman-Frankel method with 2,4-dinitrophenylhydrazine. The method of SDH activity measurement was based on the catalytic reduction of fructose to sorbitol utilizing the coenzyme NADH. Assay of BUN was based on two coupled enzymatic reactions in which ammonia released from urea was determined.

Determination of Toluidine Isomers in Urine

The method involved the isolation of toluidines with toluene, derivatisation with heptafluorobutyric anhydride (HFBA) and capillary gas chromatographic analysis (Jodynis-Liebert and Bennasir 2000).

Statistical Analysis

All data were expressed as the mean ±S D. Treated groups were compared with respective controls and additionaly parallel groups receiving different kinds of diet were compared. Analysis of variance and Student t test was used. If data did not show homogeneity of variances, Kruskal-Wallis test followed by multiple comparisons Newman-Keuls test were performed.

RESULTS

Food and water consumption in controls and treated animals showed no significant differences. No remarkable changes in general appearance of rats were observed. Body weight gains of rats during the 3-month experiment are presented in Table 2. Data from 1-month treatment was very similar, hence, they were not shown. There was not a clear relationship between the treatment with toluidines and body weight gain of the rats; however, the lowest body weight gain was noted in animals treated with the higher doses of o- and p-toluidine. In some parallel groups, especially those treated with 80 mg/kg and 160 mg/kg of toluidines, body weight gain was lower in rats fed high-fat diet (Table 2).

Methemoglobin

All toluidine isomers caused statistically significant elevations in methemoglobin levels but to a different extent. Generally, a dose-response relationship was observed for all isomers in both the 1- and 3-month exposure periods. In groups treated with two higher doses of o-toluidine the range of MetHb level increase was 1.7- to 3.9-fold after 1-month treatment (Table 3). Prolonged exposure to this isomer resulted in a significant increase in the methemoglobin level, 3.8- to 9.8-fold after 3 months. The concentration of MetHb was higher in the majority of groups fed high-fat diet, including control groups. Response of hemoglobin to m-toluidine in 1-month treatment was similar to that of o-toluidine. The magnitude of increase was 1.5- to 3.7-fold and was statistically significant at all doses with the high-fat diet. The longer-term exposure to the meta isomer did not result in the higher level of MetHb. In all groups treated with m-toluidine, the level of MetHb was slightly but significantly higher in rats fed high-fat diet. In the majority of groups treated with p-toluidine, the highest level of methemoglobin was produced. In both experimental periods, almost all groups fed high-fat diet containing the para isomer showed the lower MetHb level in comparison with the parallel groups maintained on standard diet. During 1-month treatment, the increase in MetHb level in groups fed standard diet ranged from 3.1- to 6.3-fold for the para isomer, whereas in the high-fat diet groups the increase was 2.1- to 4.1-fold. In 3-month p-toluidine exposure groups, the magnitude of changes was enhanced particularly in rats fed standard diet (4.6- to 9.4-fold) versus animals receiving high-fat diet (2.7- to 5.1-fold).

Glutathione

Mean GSH levels in the liver were significantly increased in all treated groups irrespective of the kind of diet and the period of exposure (Table 4). o-Toluidine and m-toluidine caused 2.3-to 5.3-fold increase in GSH level. p-Toluidine effect was slightly weaker, the increase ranged from 1.9- to 3.3-fold. In all groups treated with the highest doses of three isomers, the increase in the level of GSH was lower in rats fed high-fat diet; i.e., for o-toluidine 4.0-fold versus 2.3-fold (1-month) and 3.2-fold versus 2.6-fold (3-month); for m-toluidine 5.3-fold versus 2.5-fold (1-month) and 5.0-fold versus 1.9 fold (3-month); for p-toluidine 3.3-fold versus 1.9-fold (1-month) and 3.2-fold versus 1.9-fold (3-month) for the standard versus high-fat diet, respectively.

Lipid Peroxidation

The majority of animals administered ortho toluidine showed 1.5- to 4.2-fold increase in hepatic TBARS level in the absence of a dose response. m-Toluidine did not cause any change in TBARS level in the 1-month experiment, but prolonged exposure resulted in 2- to 3-fold increase in TBARS with no dose-response. The effect of p-toluidine was the most pronounced in all groups, whereby a 1.2- to 4.5-fold increase in TBARS level was observed. Generally, there was no effect of diet except for p-toluidine where the increase in the mean level of hepatic TBARS in high-fat diet groups was lower, about 1.2- and 2.6-fold, than in the rats fed standard diet, 2.6- to 4.5-fold. A similar relationship with respect to diet was observed in rats administered the highest dose of o-toluidine for both experimental periods (Table 5).

Alanine Aminotransferase

Alterations in serum AST and SDH activities were insignificant, hence the data were not shown. ALT activity in serum of rats treated with o- and p-toluidine is presented in Table 6. m-Toluidine did not cause any changes in ALT activity (data not shown). The ALT activity was slightly increased,1.4- to 1.9-fold, only in standard diet groups treated with o-toluidine during 1 and 3 months. p-Toluidine caused a slight increase in ALT activity (1.3- to 1.8-fold) in the majority of treated groups irrespective of the kind of diet and the period of exposure.

Blood Urea Nitrogen

The most pronounced elevation in BUN level was observed in rats administered with o-toluidine during 3-month experiment, 2- to 6-fold, irrespective of the kind of diet. m-Toluidine treatment for 1 month caused the increase in BUN level which was slightly higher in standard diet groups, 2.6- to 3.9-fold, than in high-fat groups, 2- to 3-fold. In 3-month exposure to m-toluidine a slight, 1.7- to 1.9-fold, increase in BUN concentration in high-fat diet groups was observed only in the highest dose groups (Table 7). p-Toluidine did not affect BUN level (data not shown).

Urinary Content of Toluidines

In 3-month experiments urine was collected every 2 weeks for toluidines determination. To avoid too extensive diagrams only 6-, 8-, 10-, and 12-week data were presented in figures. Results for 2- and 4-week time points were similar to those in 1-month exposure.

The amounts of o-toluidine found in 24-h urine of rats in 1-month exposure ranged from 0.6% to 7.6% of the daily dose in rats fed standard diet and 0.6% to 2.3% in rats fed high-fat diet. Relationship between the dose and the amount excreted was observed only in 1- and 4-week time points (Figure 1). The amounts of o-toluidine in urine of rats in 3-month experiment were lower than in 1-month exposure, 0.3% to 2.4%, irrespective of the kind of diet (Figure 2). In general, lower amounts of o-toluidine were excreted in rats fed high-fat diet, however; statistically significant difference (p < .05) was observed only at certain doses in four time points (weeks 3, 4, 6, and 8). No clear relationship between dose and amount of o-toluidine in urine was observed.

Distinctly greater amounts of m-toluidine were excreted in urine. In 1-month experiment the range of dose percentage was 3.8% to 12.6% for rats fed standard diet and 3.2% to 12.3% for rats receiving high-fat diet. In the majority of time points there was less m-toluidine in the urine of rats fed high-fat diet compared with the standard diet group (Figure 3). In 3-month exposure the amount of excreted compound was markedly lower both in standard diet groups, 2.4% to 6.7%, and in high-fat diet groups, 2.1% to 6.2%, compared to 1-month exposure. Out of six time points tested in 3-month experiment, only in the 6-week time point a clear dose–amount excreted relationship and the significant difference between amounts excreted in two diet groups were observed (Figure 4).

The amount of p-toluidine excreted in 24-h urine during 1-month exposure was lower than those of m-toluidine; the range of percentage of the dose excreted was 1.2% to 4.1%. Relationship between the dose and the amount excreted was not very clear. In several groups the urinary amount of p-toluidine was higher in rats fed high-fat diet The difference was not statistically significant except for the 160 mg/kg group at 4 weeks (Figure 5). In 3-month experiment the amount of p-toluidine ranged from 1.2% to 5.7% of the dose in animals fed standard diet and from 1.6% to 6.1% in groups receiving high-fat diet. In the majority of groups the urinary amount of p-toluidine was lower in rats fed high-fat diet; however, only in four groups this difference was statistically significant (Figure 6).

DISCUSSION

Methemoglobin level is a single parameter in which a distinct dose-response relationship was observed, especially for o- and m-toluidine. The period of exposure also affected the MetHb formation because MetHb content was higher in rats treated with o- and m-toluidine for 3 months in comparison with those after 1 month of exposure.

Dose-response relationship was also reported for another methemoglobin-forming compound, trinitrobenzene, during subchronic (90 days) (Reddy et al. 1998) and chronic (2 years) (Reddy et al. 2000) feeding studies in rats.

The only subchronic experiment in which methemoglobingenic properties of toluidines in rats were tested was reported by Malik-Bryś and Seńczuk (1995). In 6-month exposition (to the same doses as in our study) the level of MetHb was even lower for o- and p-toluidine, 6% and 7.5%, respectively than those from our 3-month experiment, about 13%. It could be suggested that some adaptive mechanisms are involved in hemoglobin response to toluidines during long-term exposure.

In untreated animals the level of MetHb was the same in both diet groups. Some differences occured in treated rats. In general, o- and m-toluidine in both exposure periods caused a greater increase in MetHb level in rats fed high-fat diet. The effect of p-toluidine was the opposite—the level of MetHb in rats fed high-fat diet was significantly lower than that in standard diet groups.

The explanation for this opposite effect would be results reported by Gnojkowski et al. (1984). The authors showed that p-toluidine was the only isomer that induced the activity of enzymes responsible for detoxication, epoxide hydrolase and glutathione S-transferase, in the liver of rats. Moreover, only p-toluidine caused the decrease in cytochrome P450 content and in the activity of aryl hydrocarbon hydroxylase and aminopyrine N-demethylase, i.e., enzymes that can potentially catalyze the production of active metabolites. Hence, it could be assumed that the production of toxic metabolites of p-toluidine responsible for methemoglobingenic activity was lower and simultaneously the detoxification of these metabolites was enhanced in comparison with the other isomers. Probably in our experiment such effect was demonstrated in rats fed high-fat diet because polyunsaturated fatty acids induce phase II enzymes such as epoxide hydrolase and glutathione S-transferase (Yang et al. 1993; Gower 1988).

Many compounds decrease the GSH level in tissues by formation of adducts with GSH or by its oxidation to GSSG. Classical hepatotoxins such as carbon tetrachloride, bromobenzene, or acetaminophen cause a very significant GSH depletion soon after administration (Comporti et al. 1991; Szymańska et al. 1992; Özdemirler et al. 1994; Singh, Khanna, and Chander 1999; Summer et al. 1996). After the phase of GSH deficiency, its level increases and usually a hypercompensation is observed. This is probably the reason why GSH depletion is only seen following a single high dose of a chemical; repeated exposure studies usually fail to reveal GSH deficit (Parke and Piotrowski 1996). This is the case in the present study. It was shown that GSH level in treated animals was 1.2- to 5.3-fold higher than in controls.

In general, no association between the GSH level and the kind of diet, and no clear dose-response relationship were observed in the present study. Indeed, statistically significant differences were shown between the majority of the parallel diet groups, but they were rather small and inconsistent. The only consistency was the lower level of GSH observed in all the highest dose groups fed high-fat diet. A single report concerning the effect of high-fat diet on GSH level was found in available literature. Kuralay et al. (1988) showed that the hepatic GSH content in rats fed a diet supplemented by 8% fish oil was increased about 2.5-fold but such an effect was not observed when the diet contained 8% corn oil. Sunflower oil used in the present experiment did not cause any changes in GSH level as well, which could be explained by similar content of PUFAs in both kinds of oils (sunflower and corn)—much lower than in fish oil.

The increase in hepatic lipid peroxidation was observed in the majority of treated groups. It has been suggested that free radicals produced as a result of o-toluidine biotransformation could be involved in lipid peroxidation process (Ohkuma et al. 1999). It cannot be excluded that two other isomers also stimulate the generation of free radicals, which is reflected by the enhancement of lipid peroxidation. As mentioned above, PUFAs alter many functions of the biological membrane and can change membrane characteristics, including susceptibility to peroxidation. Generally, membranes that are high in PUFAs are more susceptible to peroxidation and require greater antioxidant protection in response to an oxidative stress (Murphy 1990; Gower 1988). Some of our findings are conflicting with this commonly accepted view. In our experiment no clear relationship between the content of dietary fat and the level of TBARS in the liver was observed. High-fat diet caused the higher level of TBARS only in singly treated groups. Results presented by Kuratko, Tsai, and Pence (1994) are similar to our findings. The authors fed rats three types of diet containing 20% menhaden oil or 20% corn oil or 20% beef tallow for 9 months and no diet-dependent changes in noninduced lipid peroxidation were found. In our experiment also no difference in TBARS level between untreated rats fed standard and high-fat diet was observed.

An increase in serum activities of ALT, AST, and SDH considered liver specific enzymes in rats are used as markers of hepatocellular necrosis or increased membrane permeability. A highly marked elevation of these enzymes activity is usually observed in acute intoxication with compounds damaging the liver, e.g., carbon tetrachloride (Singh, Khanna, and Chander 1999), acetaminophen (Özdemirler et al. 1994), bromobenzene (Szymańska 1996), aflatoxin B₁ (Souza, Tom, and Rao 1999). Determination of these enzymes activity was also recommended for long-term experiments (Travlos et al. 1996). In our studies ALT was the only hepatic enzyme which activity was slightly affected by o- and p-toluidine. However, no association between the enzyme activity and the kind of diet as well as no dose-response relationship were observed. Hence, because other hepatic enzymes in serum were not affected, it could be considered that toluidines did not show hepatic specific toxicity in the conditions of the experiment described.

The observed changes in BUN level were not dose related. The most marked increase in BUN concentration was produced by o-toluidine—this effect was enhanced in the 3-month experiment. Because the serum concentration of BUN is an accepted screening test for kidney function, it can be concluded that o-toluidine is the only isomer which disturbed renal function in the present experiment. Analysis of the data showed no association between the changes in BUN level and the type of diet.

Cheever, Richards, and Plotnick (1980) found that there were no major differences in the metabolism of the toluidine isomers, which would explain carcinogenic activity elicited exclusively by the ortho isomer. They postulated that formation of tumors in the urinary bladder could be associated with a higher urinary concentration of o-toluidine in comparison with those of the other isomers. Determination of toluidines content in urine was undertaken to verify this hypothesis and to examine the effect of dietary fat on uchanged toluidines excretion.

It is generally accepted that a high content of fat in diet can facilitate the absorption of lipophilic substances. It could be expected that o- and p-toluidine, which are not soluble in water, were better absorbed with high-fat diet and their amounts in urine were increased. The presented data did not support such expectation, because the excretion of o- and m-toluidine in rats fed a high-fat diet was lower in comparison with standard diet group. p-Toluidine excretion was affected by the dietary fat to a less degree.

Clinton and Visek (1989) studied the effects of diet containing 25% corn oil on the tissue distribution and excretion of ¹⁴C-7,12-dimethylbenz[a]antracene (DMBA) in rats. They did not observe any differences in cumulative urinary and fecal excretion of the compound between standard and high-fat diet. However, the highest tissue concentration was found in rats fed a high-fat diet prior to DMBA administration. It is probable that toluidines were also better absorbed in animals fed a high-fat diet, although, similarly as in DMBA study, this was not reflected by their content in urine. However, it cannot be excluded that their concentrations in tissues were increased by dietary fat.

The amount of unchanged compound in urine depends also on its biotransformation. It could be speculated that the lower amounts of o-toluidine and m-toluidine in urine of rats fed high-fat diet were due to enhanced biotransformation of these isomers. This suggestion could be confirmed by the findings of Gnojkowski et al. (1984) at least with respect to o-toluidine, the only isomer that induced some phase I drug-metabolizing enzymes. As mentioned before, the induction of these enzymes might have been additionally enhanced by dietary fat (Ioannides 1999). Our results did not confirme the hypothesis of Cheever, Richards, and Plotnick (1980) because the amounts of o-toluidine in urine was the lowest in comparison with other isomers. However, if assume that the low amount of o-toluidine in urine was due to its enhanced biotransformation and that active metabolites of this compound are resposible for its carcinogenic activity, it could be concluded that the high-fat diet potentiates o-toluidine toxicity. There is certain consistency between the highest amount of m-toluidine in 24-h urine and the weakest methemoglobin-forming properties. It might be due to the minor, in comparison with other isomers, biotransformation, i.e., production of smaller amount of metabolites responsible for MetHb formation.

The amount of toluidines in urine after repeated administration with a diet did not refer to the amounts found in urine after single oral dose administration. Seńczuk and Rucińska (1984b) reported that 25% of o-toluidine and 10% of the meta and para isomers were excreted in 24-h urine. Cheever, Richards, and Plotnick (1980) found that 21% of single dose of o-toluidine and about 2.5% of m- and p-toluidine were excreted in urine. In contrary to above finding m-toluidine amounts in 24-h urine were the highest in the present studies.

In summary, in the conditions of subchronic exposure m-toluidine appeared to be less potent in comparison with two other isomers as evidenced by the less pronounced effect on MetHb formation and on hepatic lipid peroxidation, by the lack of effect on serum ALT, as well as by the highest urinary content.

The higher level of dietary fat did not alter the majority of biochemical parameters assayed in rats fed toluidine isomers. The only diet-related differences were the enhancement of MetHb production by the ortho and meta isomers and the decreased amount of all isomers excreted in urine. It could be hypothesized that the latter effect was a result of enhanced biotransformation that may leads to a greater production of toxic metabolites.