Comparative Gavage Subchronic Toxicity Studies of o-Chloroaniline and m-Chloroaniline in F344 Rats and B6C3F1 Mice

M. R. Hejtmancik*,1, B. A. Trela*,2, P. J. Kurtz*, R. L. Persing*, M. J. Ryan*, J. T. Yarrington* and R. S. Chhabra{dagger}

* Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201; and {dagger} Division of Toxicology Research and Testing Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received January 4, 2002; accepted April 30, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ortho-Chloroaniline (o-CA) andmeta-chloroaniline (m-CA) are chemical intermediates for pigment production in the textile industry. Comparative subchronic gavage studies were conducted to determine the effect of structure on toxicity.o-CA orm-CA was administered to 10 animals/sex/species in deionized water at dosages of 0, 10, 20, 40, 80, and 160 mg/kg for 13 weeks. Blood samples for clinical pathology were collected after 3 and 23 days in rats and at study termination (Day 93) in rats and mice. No mortalities occurred that could be directly attributed to treatment. Transient clinical signs of toxicity observed after dosing included cyanosis in rats and ataxia and tremors in mice. Methemoglobin formation was directly related to dosage (rats and mice) and duration of treatment (rats). At study termination, Heinz body formation in erythrocytes in association with decreased hemoglobin, hematocrit, and red blood cell count was a prominent treatment-related effect. Enlarged spleens (gross necropsy observation) and increased spleen weight were treatment effects of each chemical in both species. Microscopic lesions typical of increased red blood cell production were found in hematopoietic tissues (bone marrow, spleen, and liver), while lesions due to increased red cell destruction were found in these tissues and also the kidneys (rats). Microscopic changes were more frequently seen and severe, and involved more body organs, in rats than mice, and in m-CA-treated animals thano-CA-treated animals. Sex differences in lesion incidence/severity were not evident.

Key Words: o-chloroaniline; p-chloroaniline; methemoglobin; Heinz body formation; hematopoiesis; splenomegaly; F344 rats; B6C3F1 mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ortho-Chloroaniline (o-CA) and meta-chloroaniline (m-CA) are chemical intermediates for the production of azo dyes, petroleum and rubber products, pharmaceuticals, and pesticides (IARC, 1982Go). This pattern of use can result in worker exposure in the chemical, dye, and agricultural industries. The chloroanilines were recommended for evaluation by the National Cancer Institute due to a high incidence of bladder cancer in dye manufacturing industrial workers (Anthony and Thomas, 1970Go). Chloroanilines are released into the environment through the metabolism or degradation of herbicides used in agriculture (Hargesheimer et al., 1981Go), and residues present in food can act as contaminants (HSDB, 2000Go). While the toxicity of aniline and p-CA has been elucidated, little information is available on the toxicity of o-CA and m-CA, in spite of widespread industrial use and consequent human exposure.

A major toxic affect of aniline (Kiese, 1966Go) and its chlorinated derivatives (Chhabra et al., 1990Go; McLean et al., 1969Go) is the production of methemoglobin in erythrocytes, resulting from the oxidation of heme iron from the ferrous to ferric state. Methemoglobin is physiologically inactive and cannot bind reversibly with oxygen. Since the normal cooperativity between heme groups that is required to facilitate the loading and unloading of oxygen is compromised, the normal oxyhemoglobin dissociation curve is shifted to the left and a smaller proportion of blood oxygen is available for release into tissues. Circulating levels of methemoglobin have been shown to produce a greater impairment in peripheral oxygen transport than an equivalent true anemia produced by an actual reduction in the red blood cell count (Darling and Roughton, 1942Go). Methemoglobinemia is usually a transient effect due to intraerythrocytic mechanisms that facilitate the conversion of methemoglobin back to hemoglobin, but it may be sustained upon repeated chemical exposure. The resulting generalized hypoxia may lead to secondary central nervous system and cardiac disorders. In addition to methemoglobin formation, rats and mice exposed to p-CA for 13 consecutive weeks showed hemolytic anemia, splenomegaly, and extramedullary hematopoiesis (Chhabra et al., 1990Go). These studies suggest that the hematopoietic system is the primary target organ of chloroaniline toxicity and that secondary effects resulting from erythrocyte damage or destruction can occur with repeated chemical exposure.

Few studies have been performed in laboratory animals to examine the toxicity of o-CA and m-CA. The oral LD50 values for both o- and m-CA (RTEC, 1993Go) have been reported to be 256 mg/kg in rats and 334 mg/kg in mice and 300 mg/kg in rats for p-CA (Symth et al., 1962Go), suggesting equal potency upon acute administration. In cats, a differential methemoglobin response was demonstrated between these three isomers, suggesting toxicity may differ with prolonged administration (McLean et al., 1969Go).

Toxicity studies were conducted with o-CA and m-CA to determine a structure-activity relationship among these isomers. Thirteen-week studies were conducted in rats and mice to determine similarities and differences in sensitivity in toxicity between rats and mice and between sexes (NTP, 1998Go). Previous studies (Chhabra et al., 1990Go) had been conducted with p-CA and results were available for comparison. Due to the hematological abnormalities reported for aniline and its chlorinated derivatives, extra rats were added to the experimental design of these comparative subchronic studies for the periodic assessment of hematological effects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Technical grade o-CA (CAS No. 95-51-2) and m-CA (CAS No. 108-42-9) were obtained in single lots from Aldrich Chemical Company (Milwaukee, WI). The chemicals were approximately 99.8% pure based on their gas chromatographic analyses. Periodic bulk chemical reanalyses by HPLC revealed comparable purity results and indicated no degradation during storage at room temperature (~25°C) protected from light. Doses were formulated for oral gavage in deionized water containing 0.1 N hydrochloric acid (pH ~2). The dose concentrations for this study were 0, 1, 2, 4, 8, and 16 mg/ml for mice and 0, 2, 4, 8, 16, and 32 mg/ml for rats. Prior to the study, a 35-day stability was established for a 1-mg/ml solution, which was the lowest concentration administered. Gavage dosing solutions were prepared once every 4 weeks, placed in amber glass bottles, and were refrigerated until used for dosing. All preadministration dose analyses indicated concentrations were within 10% of the target concentration. A mid-study postadministration dose analysis agreed with preadministration values indicating no decomposition or degradation occurred during animal treatment.

Animals.
All animals were obtained from Charles River Breeding Laboratories (Raleigh, NC). F344 rats and B6C3F1 mice were shipped at approximately 5 weeks of age, were quarantined for 10–14 days, and were approximately 7-weeks-old at the start of the study. Animals were randomly assigned to dose groups by sex and body weight by partitioning algorithm using a Xybion® computer program. There were no statistically significant differences between group mean body weights prior to initiation of these studies. Rats were housed 5/cage and mice were individually housed in polycarbonate cages with hardwood chips as bedding. NIH-07 feed and tap water were available ad libitum. The animal room temperatures were approximately 72 ± 3°F and humidity was 50 ± 15%. Fluorescent lights were on for 12 h/day, and there were a minimum of 10 room air changes per h.

Experimental design.
Groups of 10 animals of each species and sex were given o-CA or m-CA by gavage at doses of 0, 10, 20, 40, 80, and 160 mg/kg body weight, once a day, 5 days/week, for 13 weeks. Extra rats (10 sex/dose group) were included for clinical pathology evaluations that were performed on study days 3 and 23; after the second blood collection, these animals were terminated and discarded without examination. The volume of vehicle and dose solution administered was 5 ml/kg for rats and 10 ml/kg for mice. Animals were checked twice each day for signs for moribundity or morbidity, and were examined once weekly for clinical signs of toxicity. The individual animals body weights were recorded once weekly, and the most recent weight was used to determine the dosing volume. At study termination, survivors were weighed, anesthetized with carbon dioxide, and were bled for clinical pathology studies prior to euthanasia and necropsy. Animals received at least two consecutive dose administrations, with the last dose administered approximately 30 min prior to bleeding. Blood was collected into microcollection tubes (Sarstedt, Inc., Newton, NC) containing potassium-EDTA for hematology studies and into serum separator tubes to obtain samples for clinical chemistry. Clinical chemistry parameters included urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts. Hematological analyses were performed with a Serono-Baker System 9000 Hematology Analyzer, and serum clinical chemistry determinations were performed using a Hitachi 704 Chemistry Analyzer. Methemoglobin concentrations were determined by the spectrophotometric method of Evelyn and Malloy (1938)

All animals, including early death animals, received a necropsy. During each necropsy, all tissues were examined in situ for gross lesions. At study termination, selected organ weights (spleen, liver, thymus, heart, lung, and right testis and kidney) were determined. All collected tissues were preserved in 10% neutral buffered formalin. Tissue examined microscopically in all control, high dose, and early death animals included adrenal glands, brain, clitoral glands, esophagus, bone marrow (femur), gallbladder (mice), heart, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidneys, liver, lungs and mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, ovaries, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate gland, salivary glands, seminal vesicles, spinal cord, spleen, stomach, testis (with epididymis), thymus, thyroid gland, trachea, urinary bladder, uterus, and any gross lesions seen at necropsy. In rats, the bone marrow, kidney, spleen, and liver were identified as target organs and examined in lower dose animals. In mice, the spleen, bone marrow, and liver (m-CA only) were identified as targets and examined at lower doses. The severity of lesions was graded from minimal to marked on a 1–4 scale.

In-life data (body weights, clinical observations) and microscopic findings were collected and summarized using a computerized system (Toxicology Data Management System) provided by the National Toxicology Program. Body weight and organ weight data were analyzed using the parametric comparison procedures of (Williams, 1971Go, Williams, 1972Go) or Dunnett (1955)Go. Clinical pathology data were analyzed using the nonparametric comparative procedures of Shirley (1977) or Dunn (1964). The Fisher exact test, a procedure based on the overall proportion of affected animals, was used to analyze histopathology findings (Gart et al., 1979Go). On all tables (unless indicated otherwise), values are expressed as group mean and SE. Significant differences from control are indicated by asterisks (*p <= 0.05, **p <= 0.01).


    RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The survival and group mean body weights at the termination of these subchronic studies are included in Table 1Go for rats and Table 2Go for mice. The cause of death of three treated rats that were found dead over the course of this study was undetermined. Survival was similar between rat treatment groups receiving equivalent doses of o-CA or m-CA and control groups. o-CA treatment produced a small but statistically significant depression in group mean body weight of 6.7% in the male rat 160 mg/kg dose group. m-CA treatment produced a 10.5% depression in group mean body weight relative to control in the 160 mg/kg male rat dose group. Six early deaths occurred in the o-CA mice study and 9 in the m-CA mice study. These deaths were distributed equally across control and treated groups and could not be attributed to chemical treatment at necropsy. For each chemical, group mean body weights of the mice treatment groups at study termination were similar to that of control, except for an 8% decrease in high dose o-CA female mice.


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TABLE 1 Survival and Mean Body Weights in Rats for the 13-Week Gavage Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 

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TABLE 2 Survival and Mean Body Weights in Mice for the 13-Week Gavage Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 
Spleen weight to body weight ratios are shown in Table 3Go. Treatment produced a dose-related increase in relative spleen weight. At each dose level, the increase was greater in animals given m-CA. A similar trend occurred in absolute spleen weight (data not shown), indicating relative weight increases were not due to body weight depressions. The weights of liver, thymus, kidney, testes, and lung for dosed groups were not significantly different from those of vehicle controls (data not shown). The only other treatment related change was a slight increase in relative heart weight in rats given 80 and/or 160 mg/kg o-CA or m-CA or in mice given 160 mg/kg o-CA (Table 4Go).


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TABLE 3 Relative Spleen Weights in Rats and Mice for the 13-Week Gavage Studies of o-Chloroaniline (o-CA) andm-Chloroaniline (m-CA)
 

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TABLE 4 Relative Heart Weights in Rats and Mice for the 13-Week Gavage Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 
Methemoglobin concentrations in rats are included in Table 5Go. Both chemicals produced a dose-related increase in methemoglobin concentrations. The formation of methemoglobin was an early manifestation of chloroaniline exposure. After the administration of only three doses, a dose-related increase in blood methemoglobin concentration was detected in all m-CA treatment groups. Early elevations in methemoglobin concentrations occurred in the o-CA treatment groups, but only in the higher dose groups (>=40 mg/kg for females and >=80 mg/kg for males). At Days 23 and 93, the methemoglobin increase was also greater in rats given m-CA. In rats given o-CA, the magnitude of change increased in mean methemoglobin concentration in all dose groups over the duration of the study, with a clear dose-related trend occurring by Day 93. A similar progressive increase in blood methemoglobin concentration occurred with prolonged exposure time in male rats given m-CA at doses up to 80 mg/kg and in female rats given m-CA up to 40 mg/kg. Male rats given 160 mg/kg and female rats given 80 and 160 mg/kg m-CA showed high and relatively constant (sustained) methemoglobin concentrations at each sampling time, suggesting a maximal response had been achieved. At each dose, the methemoglobin response was generally greater in female rats. Methemoglobin concentrations in mice at study termination are included in Table 6Go. Again, treatment was associated with a clear dose-related increase in blood methemoglobin concentrations and this response was greater at equivalent dose levels in mice given m-CA compared to o-CA. Methemoglobin concentrations were generally higher in rats than mice. At each dose, the methemoglobin response was generally greater in female mice when compared to male mice.


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TABLE 5 Methemoglobin Concentrations in Rats for the 13-Week Gavage Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 

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TABLE 6 Methemoglobin Concentrations in Mice Administered o-Chloroaniline (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 
Clinical signs of toxicity followed dosing with both chemicals. Approximately 30 min following gavage administration, rats in the higher dose groups (80 and 160 mg/kg) were found to have darker eyes (iris) than control and developed a blue discoloration of the skin in areas easily observed (ear, nasal, genital, and footpad regions). These effects were attributed to anoxia resulting from the formation of high levels of methemoglobin. Clinical signs were also observed in high dose (160 mg/kg) mice and consisted of slight to mild tremors and ataxia that occurred almost immediately after dosing and persisted for approximately 15 min. These neurological signs were attributed to chemically induced cyanosis due to methemoglobin formation.

Erythrocyte counts of animals at study termination are included in Table 7Go. Exposure to either chemical produced dose-related decreases in erythrocyte count, which were statistically significant at the higher doses. For both o-CA and m-CA, female rats were affected at lower doses than males. At equivalent dose levels, m-CA generally was more potent than o-CA.


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TABLE 7 Erythrocyte Counts in Rats and Mice Administered o-Chloroaniline (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 
The percentages of erythrocytes containing Heinz bodies are shown in Table 8Go. With o-CA, only high dose (160 mg/kg) rats and mice showed a significant increase in the number of erythrocytes with Heinz bodies. With m-CA, rats at the 40 mg/kg dose level and above and mice at the 80 and 160 mg/kg dose levels showed significant and dose-related (except 160 mg/kg m-CA rats) increases. Other findings with either chemical included polychromasia and poikilocytosis (presence of erythrocytes with abnormal shapes).


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TABLE 8 Percentage of Erythrocyte-Containing Heinz Bodies in Rats and Mice Administered o-Chloroaniline (o-CA) OR m-Chloroaniline (m-CA) for 93 Days
 
Anemia was associated with a decrease in hematocrit in rats and mice (Table 9Go) and a decrease in hemoglobin concentration in rats (Table 10Go). The hemoglobin concentration was not a reliable predictor of anemia in mice receiving the higher doses of o-CA or m-CA or female mice receiving m-CA. The hemoglobin concentrations for these groups were either unchanged or slightly increased relative to the changes in hematocrit or erythrocyte count. This paradoxical response is consistent with the erroneous elevation of the hemoglobin concentration due to the presence of large numbers of Heinz bodies. Mean corpuscular volume was not affected (data not shown) indicating the anemia was normocytic. A slight increase in mean corpuscular hemoglobin or in mean corpuscular hemoglobin in high dose animals (data not shown) indicates the anemia was hyperchromic. Again, this response is consistent with either a hemolytic process or the presence of large numbers of Heinz bodies producing erroneously elevated hemoglobin concentrations. Reticulocytes were elevated in rats and mice at the higher dosages of m-CA or o-CA (Table 11Go), and these increases are consistent with an erythropoietic response to anemia.


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TABLE 9 Hematocrit in Rats and Mice Administered o-Chloroaniline (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 

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TABLE 10 Hemoglobin Concentration in Rats and Mice Administered o-ChloroanilinE (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 

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TABLE 11 Reticulocyte Counts in Rats and Mice Administered o-Chloroaniline (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 
Splenic abnormalities were observed at necropsy and are included in Table 12Go. Splenomegaly was observed in both rat sexes at a lower dose of m-CA (40 mg/kg) than o-CA (80 mg/kg). In mice, splenomegaly was observed only with m-CA at the high dose levels. While no enlargement occurred with o-CA, the spleens of high dose mice were abnormal (discolored) in appearance.


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TABLE 12 Incidence of Enlarged Spleens at Necropsy in Rats and Mice Administered o-Chloroaniline (o-CA) or m-Chloroaniline (m-CA) for 93 Days
 
Microscopic lesions consisted of excess hematopoietic activity in the bone marrow, spleen and/or liver, and deposition of hemosiderin pigment in the bone marrow, spleen, liver (Kupffer cells), or kidney cortex (rats). These changes were seen more frequently and/or severely in rats (Table 13Go) than mice (Table 14Go), and more frequently/extensively in m-CA-treated animals than o-CA-treated animals. Sex differences were not evident in lesion frequency/severity. Splenic vascular congestion and cellular infiltration/fibrosis of the splenic capsule were microscopic lesions found only in rats (both sexes). Splenic congestion was found only in m-CA-treated rats, while cellular infiltration/fibrosis was found in m-CA- and o-CA-treated rats.


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TABLE 13 Incidence and Average Severity of Treatment-Related Lesions in Rats in the 13-Week Gavage Toxicity Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 

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TABLE 14 Incidence and Average Severity of Treatment-Related Lesions in Mice in the 13-Week Gavage Toxicity Studies of o-Chloroaniline (o-CA) and m-Chloroaniline (m-CA)
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pattern of toxic effects produced by o- and m-chloroaniline was similar in the two species studied. As reported for aniline (Kiese, 1966Go) and its chlorinated derivatives (Chhabra et al., 1990Go; McLean et al., 1969Go), the hematopoietic system was primarily affected. The predominant manifestation of toxicity was the production of methemoglobin and Heinz bodies that produced red blood cell abnormalities. Acute methemoglobinemia may be life-threatening when the level of methemoglobin exceeds half of the total circulating hemoglobin (Beutler, 1972Go). While some mild oxidants such as nitrite produce only methemoglobin formation, strong oxidants produce methemoglobin in conjunction with Heinz body formation. Heinz bodies are dense bodies of denatured hemoglobin in erythrocytes. Their occurrence can produce gross distortions in shape of red blood cells, resulting in premature splenic phagocytosis, or impairment of ionic transport that may cause changes in osmotic pressure, hyperpermeability, and intravascular hemolysis. Anemia associated with Heinz body formation is typically hemolytic and is followed by a compensatory increase in erythropoiesis. In this study, a dose-related increase in spleen weight was observed for both rats and mice. A similar enlargement of the spleen has been reported to occur in rats dosed with aniline (Gralla et al., 1979Go) and in rats and mice dosed with p-CA (Chhabra et al., 1990Go). Splenomegaly was attributed to sequestration of damaged erythrocytes in splenic sinusoids and to a compensatory increased hematopoiesis. These splenic effects appeared to be secondary to erythrocyte toxicity, since there were no direct or primary microscopic abnormalities in the spleen.

At equivalent dosage levels, m-CA was more potent than o-CA in producing erythrocyte toxicity. A lower threshold dose for induction of toxicity occurred with m-CA. A striking difference was also seen in the magnitude of the methemoglobin response. A high methemoglobin concentration was found at each sampling in the m-CA high dose groups, suggesting that a sustained maximal response had been achieved. In contrast, a more gradual increase in methemoglobin occurred in the o-CA treatment groups. These results are consistent with studies of methemoglobin formation in cats (McLean et al., 1969Go). These studies showed that the m- and p-halo aniline derivatives appeared to be the most potent methemoglobin inducers. In previous studies with p-CA, significant increases in methemoglobin occurred at gavage doses lower than the doses used in the present evaluation (Chhabra et al., 1990Go). These findings suggest that the order of potency for methemoglobin formation for the chloroaniline isomers in rats and mice is p-CA > m-CA > o-CA. A similar order of potency occurred with changes in other hematology parameters, spleen weights, gross/microscopic abnormalities, and in the severity of pigment (hemosiderin) deposition. This potency order is identical to that reported for methemoglobin formation in cats for chloroaniline (McLean et al., 1969Go). Substitution at the para position increased activity, while substitution at the o- and m-position reduced activity.

Aromatic amines such as aniline only produce methemoglobin in intact animals and not in vitro when incubated with blood or red blood cell suspensions (Smith, 1996Go). An n-hydroxyl metabolite is believed to be the active intermediate. The difference in potency among the CA isomers may be related to differences in the ratios of bioactivation or inactivation of reactive intermediates (Dial et al., 1998Go).

The methemoglobin response in rats was greater in magnitude than that in mice. Methemoglobin is reduced to hemoglobin in mammalian species by an NADH-dependent methemoglobin reductase in erythrocytes. The activity of this degradative enzyme is approximately 2-fold higher in mice than in rats, which may explain the lower methemoglobin response in mice (Smith, 1996Go). Since the activity of this enzyme is relatively low in human erythrocytes, humans may be more susceptible to the toxic effects of aniline and its chlorinated derivatives.

Female rats were more susceptible to the formation of methemoglobin and the development of anemia than male rats in the o- and m-CA studies. The mechanism for this sex difference is unknown. Generally, female rats have slightly fewer but larger erythrocytes than male rats, which may predispose the erythrocytes of female rats to be more sensitive to oxidative injury. Additionally, male animals may be better able to respond to anemia due to the stimulatory effect of androgens on erythropoiesis (Jain, 1986Go).

Aniline and chlorinated derivatives have been demonstrated to have carcinogenic activity in humans and laboratory animals. An increased incidence of bladder cancer has been reported in men involved in the manufacture or use of aniline, particularly in the dye industry (IARC, 1982Go). Aniline was found to produce splenic sarcomas in male F344 rats (Goodman et al., 1984Go). p-CA was shown to produce splenic sarcomas in male rats and an increased incidence of hepatocellular neoplasms and hemangiosarcomas of the liver and spleen in male mice (Chhabra et al., 1991Go). Binding of aniline-derived radioactivity to DNA, RNA, and proteins occurs in a number of tissues in the rat, including the spleen (McCarthy et al., 1985Go). Inflammatory lesions and tumors of the spleen in rats are uncommon (Stefanski et al., 1990Go). Chemicals structurally related to aniline (azobenzene, D & C Red No. 9, dapsone, and o-toluidine) have also been shown to be carcinogens in rats (NIEHS, 1993Go). Chronic overstimulation of the hematopoietic system produced by aniline and its derivatives may play a role in the induction of splenic tumors.

Structural activity studies have been conducted to elucidate the hematotoxicity of selected compounds. In a comparative study of three methylated hydroxylamines, methemoglobin formation was the primary and critical step for ortho-derivatives leading to the formation of free radicals which produced lipid peroxidation, depletion of glutathione, and the inhibition of NADPH methemoglobin reductase and glutathione-s-transferase activities (Spooren and Evelo, 1997Go). Other studies have suggested that hemolytic damage may occur by more than one mechanism, as not all methemoglobin-forming compounds (e.g., sodium nitrite and 2-aminophenol) are hemolytic. Methemoglobin formation is the most common side effect of dapsone, which limits the effectiveness for the use of this drug for the treatment of leprosy (Coleman et al., 1996Go). Two N-hydroxyl metabolites of dapsone appear to be the direct-acting hemolytic agents and that the action of these toxic metabolites in the red cell produces premature sequestration by the spleen (Jollow et al., 1995Go).

Methemoglobinemia was a primary toxic response in those comparative subchronic studies of o- and m-CA. Other abnormalities could be explained as secondary to methemoglobin formation and subsequent increases in erythrocyte injury and turnover including anemia, red cell morphological alterations (e.g., Heinz bodies), and effects on the spleen (hemosiderin accumulation, capsular fibrosis, and increased hematopoietic cell proliferation), liver (Kupffer cell hemosiderin accumulation), and bone marrow (increased hemosiderin and hematopoietic cell proliferation).

Capsular and parenchymal fibrosis of the spleen has been proposed as a potential preneoplastic lesion for the development of spleen tumors for a number of previous chronic studies including p-chloroaniline, aniline, o-toluidine, dapsone, azobenzene, and D&C Red Dye No. 9 (Bus and Popp, 1987Go). The o- and m-CA isomers produced fibrosis in the same manner as p-CA, which is already classified as a 2B carcinogen by IARC. Since erythrotoxicity is probably the most important effect following occupational exposures, the m- and o-CA isomers should be treated, for occupational health purposes, like the p-CA isomer. This conservative approach to base industrial hygiene practices on the more potent of the isomers would minimize potential occupational exposures and avoid further testing.


    ACKNOWLEDGMENTS
 
This research was supported by NIEHS Contract No. NO1-ES-15320.


    NOTES
 
A preliminary version of this work was presented at the Society of Toxicology Meeting in Dallas in March, 1994 (see Hejtmancik et al., 1994Go).

1 To whom correspondence should be addressed. Fax: (614) 424-5263. E-mail: hejtman{at}battelle.org. Back

2 Present address: Abbott Laboratories, Abbott Park, IL 60664. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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