* Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201; and
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
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ABSTRACT |
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Key Words: o-chloroaniline; p-chloroaniline; methemoglobin; Heinz body formation; hematopoiesis; splenomegaly; F344 rats; B6C3F1 mice.
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INTRODUCTION |
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A major toxic affect of aniline (Kiese, 1966) and its chlorinated derivatives (Chhabra et al., 1990
; McLean et al., 1969
) 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, 1942
). 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., 1990
). 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, 1993) 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., 1962
), 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., 1969
).
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, 1998). Previous studies (Chhabra et al., 1990
) 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.
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MATERIALS AND METHODS |
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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 1014 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 14 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, 1971, Williams, 1972
) or Dunnett (1955)
. 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., 1979
). 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).
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RESULTS |
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Erythrocyte counts of animals at study termination are included in Table 7. 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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DISCUSSION |
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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., 1969). 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., 1990
). 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., 1969
). 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, 1996). 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., 1998
).
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, 1996). 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, 1986).
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, 1982). Aniline was found to produce splenic sarcomas in male F344 rats (Goodman et al., 1984
). 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., 1991
). 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., 1985
). Inflammatory lesions and tumors of the spleen in rats are uncommon (Stefanski et al., 1990
). 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, 1993
). 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, 1997). 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., 1996
). 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., 1995
).
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, 1987). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (614) 424-5263. E-mail: hejtman{at}battelle.org.
2 Present address: Abbott Laboratories, Abbott Park, IL 60664.
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