Subacute Inhalation Toxicity of Aniline in Rats: Analysis of Time-Dependence and Concentration-Dependence of Hematotoxic and Splenic Effects

Jürgen Pauluhn1

Institute of Toxicology, Bayer HealthCare AG, D-42096 Wuppertal, Germany

Received March 8, 2004; accepted June 1, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, thirty male Wistar rats/group were exposed nose-only to mean analytical concentrations of 9.2, 32.4, 96.5, and 274.9 mg aniline/m3 using an exposure regimen of 6 h/day, 5 days/week for 2 weeks (days 0–11), followed by a 2-week post-exposure period (up to day 28). Serial sacrifices for specialized examinations were performed on days 0, 4, 11, 14, and 28. Clinical signs of toxicity, body weights, hematology, and clinical chemistry tests, including total iron in liver and spleen, splenic lipid peroxidation, organ weights, gross and histological changes in target organs were recorded. No mortality was observed during the study. Rats exposed to 96.5 mg/m3 and above displayed cyanosis, with no apparent progression during the exposure period. The predominant manifestation of toxicity was methemoglobin formation and associated erythrocytotoxicity. The changes observed included anemia, red blood cell morphological alterations (e.g., Heinz bodies), decreased hemoglobin and hematocrit, reticulocytosis, and effects on the spleen (splenomegaly, hemosiderin accumulation, and increased hematopoietic cell proliferation), which gained significance at 96.5 and 274.9 mg/m3. With regard to increased splenic extramedullary hematopoiesis, borderline effects occurred at 32.4 mg/m3. The total content of iron in spleen homogenates increased in a concentration-dependent and time-dependent manner with increasing duration of exposure. The maximum accumulation of iron in the liver and spleen exceeded the respective control levels by {approx}60% and {approx}500%, respectively. Splenic lipid peroxidation and total iron were highly correlated (r2 = 0.93) toward the end of the exposure period. A hepatic hemosiderosis was observed at 274.9 mg/m3. Thus, in regard to erythrocytotoxicity and associated increased splenic sequestration of erythrocytes, iron accumulation and lipid peroxidation 32.4 mg/m3 constitutes the no-observed-adverse-effect concentration (NOAEC). However, spleens of the 32.4 mg/m3 exposure group exhibited a minimal increase in extramedullary hematopoiesis. Exposure to 9.2 mg/m3 was not associated with any significant effect.

Key Words: methemoglobin; bone marrow; spleen; liver; hematotoxicity; aniline; lipid peroxidation; iron; oxidative stress; inhalation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Aniline is used as chemical intermediate in a variety of chemical syntheses. The primary toxicity of aniline is characterized by methemoglobinemia (Anon., 2001Go). The increased production of methemoglobin (MetHb) in erythrocytes (ferrihemoglobin HbFe3+) results from the oxidation of heme iron from the ferrous to the ferric state (Kiese, 1974Go; McLean et al., 1969Go). Although MetHb formation is reversible and does not enhance red blood cell destruction per se, it is a component of oxidative injury to red blood cells (Jollow and McMillan, 2001Go) and is generally involved as a step in Heinz body formation (Harvey, 1989Go). MetHb is physiologically inactive and cannot bind reversibly with oxygen. Methemoglobinemia is usually a transient effect of intraerythrocytic mechanisms that facilitate the conversion of MetHb back to hemoglobin. However, extended intra-erythrocytic oxidative stress promotes depletion of the antioxidant capacity of the cell, causing dysfunction by reversible interactions between protein thiols and glutathione, which leads to binding of the hemoglobin to the erythrocyte membrane (Dickinson and Forman, 2002Go; Jarolim et al., 1990Go; Marchesi, 1985Go; Mawatari and Murakami, 2004Go). The critical role proposed for the RBC membrane skeletal proteins in controlling the morphology of this cell caused by exposure to hemolytic agents has been described in detail (Grossman, Simson, and Jollow, 1992Go). Published evidence delineates that aniline is metabolized quickly at lower doses, whereas a dose-related increase in methemoglobinemia duration was accompanied by capacity limitation of aniline elimination (Harrison and Jollow, 1987Go). Aniline is known to be bioactivated in the gastrointestinal mucosa, and especially in the liver, to become a MetHb-forming agent (Khan et al., 1998Go; Kiese, 1974Go). It exerts hematotoxicity through an active/reactive metabolite mechanism, mainly N-hydroxylated metabolites, which take part in cyclic redox systems. Other MetHb-forming metabolites, such as aminophenols, are known to be formed; however, they do not appear to play a significant role because of their rapid clearance when compared phenylhydroxylamine, which is a "dead end" pathway (Grossman and Jollow, 1986Go; Harrison and Jollow, 1987Go).

In spite of its structural simplicity, the metabolism of aniline is complex (Grossman and Jollow, 1986Go; Lenk and Sterzl, 1982Go). The primary hematological event (besides methemoglobinemia) in this and other studies with arylamines is extravascular hemolysis. Hence, sustained or recurrent damage to (or "prematurely" aged) erythrocytes by aniline metabolites leads to their increased sequestration and removal by the spleen (erythroclasia). Possible mechanisms, including the apparent selective toxicity to the spleen, have been addressed in detail elsewhere (e.g., Bus and Popp, 1987Go; Ferrali et al., 1997Go; Grossman and Jollow, 1986Go; Harrison and Jollow, 1987Go; Jenkins et al., 1972Go; Jensen and Jollow, 1991Go; Khan et al., 1995aGo, 1995bGo, 1997Go, 1998Go, 1999aGo, 1999bGo, 2000Go, 2003aGo, 2003bGo; Kiese, 1974Go). Few studies have been performed in laboratory animals to examine the subacute toxicity of aniline by the inhalation route. Its known hepatic bioactivation and kinetic features make it likely that appreciable differences in toxic potency may occur when aniline is administered orally or by inhalation exposure (Pauluhn, 2002Go). The difference in potency can be attributed to differences in the extent of bioactivation or inactivation of reactive intermediates. As detailed by Kiese (1974)Go and Akintowa (2000)Go, the magnitude of MetHb formation, its reduction back to hemoglobin by a NADH-dependent methemoglobin reductase, and the ensuing erythrocytotoxicity together constitute a multistep process, yet this effect also depends appreciably on the availability of antioxidant substrates within the intact RBC. Accordingly, for aniline, in the extrapolation across routes, dosing regimens, including the dose administered per unit of time, appear to be particularly complex because it seems likely that the resulting increased availability of plasma aniline after oral administration of larger doses may be associated with rate-limited elimination kinetics and the prolonged production of metabolites in amounts sufficient to account for sustained methemoglobinemia over a long period. This also affects the capacity of the cells to reduce MetHb. Because methemoglobinemia causes a rapidly reversible type of toxicity, the peak MetHb depends on the intermediate concentration of the most active metabolite and not on the total amount (body burden) per se. Likewise, it can be assumed that both the kind of dosing regimen and the rate of dose-delivery have a major impact on the magnitude of the MetHb concentrations produced. Thus, as demonstrated for aniline, straightforward route-to-route extrapolations are considered to be error prone when the focus is on quantitative estimations (Pauluhn, 2002Go; Pauluhn and Mohr, 2001Go). Previous work by Kim and Carson (1986)Go has shown that following inhalation exposure of rats to aniline the half-life of MetHb was as short as 75 min ({approx}400 mg/m3 for 8 or 12 h). Because of this short half-time of MetHb in rats, the exact quantification requires the sampling and processing of blood within 5 minutes after cessation of exposure. As demonstrated for the MetHb-inducing agent p-phenetidine (Pauluhn and Mohr, 2001Go), non-adherence to such a rigorous sampling protocol might produce false-negative low values of MetHb in rats. The objective of this study was to analyze the concentration-dependence and time-dependence on the induction and reversibility of hematotoxic effects and subsequent secondary hepatic and splenic changes during and after a 2-week inhalation exposure period. Based on the available published evidence, this study focused on the integration of endpoints capable of demonstrating the primary effect of aniline-induced hematotoxicity (MetHb formation), associated increased sequestration of damaged erythrocytes in the reticuloendothelial system of the spleen, splenic iron accumulation and lipid peroxidation, and possible compensatory secondary responses to increased hematopoiesis. The study described was conducted in compliance with Good Laboratory Practice (GLP) requirements (OECD, 1983Go) and EU animal welfare regulations (Council of the European Communities, 1986Go).


    Methods
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Test material. Aniline (aminobenzene) was from Merck, Darmstadt, Germany (catalog no.: 822256) and was specified to be >99% pure.

Animals, diet, and housing conditions. Specific-pathogen-free, young adult male Wistar rats of the strain Hsd Cpb:WU (SPF) were from the experimental animal breeder Harlan-Winkelmann GmbH, Borchen, Germany. Although female rats are often shown to be more susceptible to the formation of MetHb and the development of anemia than male rats (Hejtmancik et al., 2002Go), this study utilized male rats only to allow a better comparison with existing mechanistic studies. At the commencement of the study the rats were approximately 2–3 months old and their average body weight was 240 g. All animals were housed individually in polycarbonate cages containing bedding material (low-dust wood shavings) and were provided with a standard fixed-formula diet (KLIBA 3883 pellets maintenance diet for rats and mice; PROVIMI KLIBA SA, 4303 Kaiseraugst, Switzerland) and municipality tap water in drinking bottles. Feed and water was available ad libitum except during exposure. Animals were acclimated to the housing conditions (12-h light/dark cycle, temperature {approx}22°C, relative humidity 40%–60%) for at least 5 days prior to initiation of exposure to the test material.

Experimental design. Thirty male Wistar rats/group were exposed nose-only to aniline vapor in targeted concentrations of 10, 30, 90, and 270 mg/m3 using an exposure regimen of 6 h/day, 5 days/week for 2 weeks (days 0–11), followed by a 2-week post-exposure period (up to day 28). The control group was exposed to conditioned, dry air under otherwise identical conditions. Serial sacrifices for specialized examinations were performed on days 0, 4, 11, 14, and 28 to address the time-course and reversibility of changes. The study design and selection of exposure concentrations took into account the findings from previous inhalation studies with aniline using male Crl:CD rats exposed head-only to 0, 64.7, 171.4, and 331.3 mg/m3, 6 h/day 5 days/week, for 2 weeks (duPont, 1982; cited in IRIS), as well as studies with the structural analogs 4-ethoxyaniline (p-phenetidine) at 11.1, 86.2, and 882.6 mg/m3 using an exposure regimen of 6 h/day, 5 days/week for 4 weeks (Pauluhn and Mohr, 2001Go). In those studies, the most salient findings included increased spleen weights, splenic hemosiderin depositions, and erythropoiesis. The NOAEC was 11.1 mg/m3 for p-phenetidine. The selection of specific endpoints considered publications of previous studies with aniline and its structural analogs—e.g., Hejtmancik et al. (2002)Go, Khan et al. (1995aGo,bGo, 1997Go, 1998Go, 1999aGo,bGo, 2000Go; 2003a)Go, and Nair et al. (1986)Go.

Exposure technique, atmosphere generation, and characterization. Vapor atmospheres of aniline were generated under dynamic conditions using a bubbler containing liquid aniline (measures of bubbler: diameter: {approx}2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level: {approx}5 cm). These bubblers were maintained at 25°C with a digitally controlled thermostat (JULABO UC, Julabo, Seelbach, Germany). Air flows passed through the liquid aniline were controlled by a calibrated gas-metering device (digitally controlled mass flow controllers) and ranged from 0.12 l/min to 3.4 l/min in the low and high group, respectively. This atmosphere was subsequently diluted by conditioned, dry air. The total flow rate directed into the nose-only exposure chamber was 30 l/min. Rats of the control group were exposed nose-only to dry, filtered air only. The inhalation chamber had a volume of 7.6 l and was suitable to accommodate 40 rats. The air flow rate supplied into and extracted from the chamber provided a slight positive balance of air flow toward the rats' breathing zone, and this was maintained at 0.75 l/min/exposure port. Details of the nose-only exposure system used have been published elsewhere (Pauluhn, 1994Go). Nominal concentrations were calculated taking into account the actually evaporated mass of test substance (weight loss of bubbler before and after exposure) divided by the total airflow through the chamber. Determinations were made daily after each exposure.

Exposure atmospheres were characterized by using a gas chromatographic (GC) technique (Hewlett-Packard GC 5890 equipped with a flame ionization detector, split/splitless injector capillary column HP Ultra 50+, length: 30 m, ID: 0.32 mm, film thickness 0.17 µm, autosampler HP 7673, and an HP 3365 WorkStation for data processing). For GC analyses, samples were taken from the vicinity of the rats' breathing zone three times per exposure day using two impingers (in-line) filled with toluene. Between the glass bubblers and the gas-metering device (digital flow controller) a cool-trap was used to scrub volatile constituents from the sampled air. For calibrations, the test substance served as reference material. The sampling flow rate was 0.5 l/min. The sample volumes were 50, 20, 10, and 10 l/sample in the 10, 30, 90, and 270 mg/m3 group, respectively. In the last two groups, the temporal stability of concentrations of aniline in the chambers was monitored continuously using a Compur Total Hydrocarbon Analyzer (Compur, Munich, Germany). Chamber temperature and humidity were measured electronically, and mean values were approximately 23°C and <5%, respectively.

Animal exposure and parameters monitored. Animals were assigned at random to the five exposure groups using 30 male rats/group. The procedures used in this study were consistent with those called for by the OECD testing guideline no. 412 (1981). During the study, animals were observed before and after exposure for signs of toxicity. During the post-exposure period, observations were made once daily. Body weights were measured twice weekly on Mondays and Fridays and once weekly during the post-exposure period. Rectal temperatures were recorded shortly after exposure using a rectal probe for rats (Digimed H 11 thermometer; Sachs Elektronik, March, Germany) on days 0, 4, and 11 on 5 rats/group. The following hematology parameters were evaluated in 5 rats/group at all sacrifices: erythrocyte count (RBC), red cell morphology, hematocrit (HCT), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte counts (RC), percentage of erythrocytes with Heinz bodies, MetHb, platelet count, clotting time (hepatoquick), and total and differential leukocyte counts. The following serum clinical chemistry parameters were evaluated in 5 rats/group at the post-exposure sacrifices (days 14 and 28): glucose, blood urea nitrogen, creatinine, total protein, albumin, triglycerides, cholesterol, total bilirubin, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (APh), {gamma}-glutamyltranspeptidase ({gamma}-GPT), calcium, inorganic phosphorus, magnesium, sodium, potassium, and chloride. During the exposure period, MetHb was determined from blood obtained from the retro-orbital sinus immediately after cessation of exposure. For the reasons detailed elsewhere (Pauluhn and Mohr, 2001Go), this blood was processed within 5 min after collection using a hemoximeter (ABL 330, Radiometer, Copenhagen, Denmark). For blood obtained during the post-exposure period, the method described by Evelyn and Malloy (cited in Richterich and Colombo, 1978Go) using Triton X-100 as lysing agent according to Anders and Chung (1984)Go, was applied. For the remaining parameters, and for collections during the post-exposure period, blood was obtained from heart puncture during sacrifice. General hematology parameters were determined with the Hematology System ADVIA 120, Bayer Diagnostic GmbH, Fernwald, Germany. Erythrocytes with Heinz bodies were counted by light microscopy after staining with rhodanile blue, modified according to Simpson et al. (1970)Go. All serum chemistries were performed using automated, conventional methodologies.

In liver homogenates the following endpoints were determined in 5 rats/group at post-exposure period sacrifices (days 14 and 28): aminopyrin-N-demethylase (N-DEM), p-nitroanisol-N-demethylase (O-DEM), cytochrome P450 (P450), triglycerides, and total iron (at all sacrifices). In spleen homogenates the following endpoints were determined in 5 rats/group at all sacrifices: glutathione peroxidase (GPx), lipid peroxidation (LPO), total iron, and ferritin. Tissue specimens were stored frozen until use (–20°C). The GPx-activity was determined using the Calbiochem-Cellular glutathione peroxidase assay-kit (catalog no. 354104). Lipid peroxidation was determined using the Calbiochem-lipid peroxidation assay kit (catalog no. 437634). In this assay the concentration of MDA (malondialdehyde) and 4-HNE (4-hydroxy-2(E)-nonenal) were determined spectrophotometrically as markers of lipid peroxidation using 1,1,3,3-tetramethoxypropane as reference standard. Ferritin was determined by direct, competitive ELISA (kit no. 52100, CellTrend, Luckenwalde, Germany) in a 96-well plate coated with anti-rat ferritin specific for the spleen. Briefly, the minced spleen (approximately 40–80 mg) was homogenized in 20 mM Tris + 10% Triton X-100 and then centrifuged. For the ELISA assay, the supernatants were used. The standards (50 µl), negative and positive controls, and supernatants were diluted in Tris-Triton (1:100 and 1:300), and anti-rat ferritin–HRP was added. The plates were then incubated for 1 h at room temperature. The binding of the anti-rat ferritin on the plate was competitively inhibited by the ferritin present in the well. After the plates were washed, the substrate solution (tetrabenzyl benzidine) was added. The optical density was measured spectrophotometrically at 450/620 nm (MWG-Biotech, Ebersberg, Germany). For iron determinations, tissues were mineralized by wet chemical digestion using sulfuric acid and nitric acid, with the residues taken up in hydrochloric acid and diluted with water. The iron content was determined with atomic absorption spectroscopy using the iron absorption band of 248.3 nm. ICP-standard iron (1000 mg Fe/l) served as reference standard.

All animals were euthanized by complete exsanguination (heart puncture) after intraperitoneal sodium-pentobarbital injection (Narcoren, Merial GmbH, Hallbergmoos, Germany). All rats were given a gross-pathological examination. Weights were recorded for brain, liver, lung, spleen, and testes at interim sacrifices (days 0, 4, and 11) and, in addition, heart, kidneys, and thymus at post-exposure sacrifices (days 14/15 and 28). For the heart, kidneys, and thymus no appreciable time-related effects were expected to occur. Therefore, the weights of these organs were determined only at the time points where the maximum effect, including reversibility, can be evaluated. From the additional 5 rats/group that were sacrificed on day 15, selected organs and tissues (spleen, femur, sternum, liver, lung) were preserved in 10% neutral-buffered formalin or Davidson's solution (testes) for histopathology. The lung was intratracheally instilled with the fixative under 20 cm H2O pressure. Osseous tissues were first decalcified and then, as for all other organs, embedded in Paraplast. All slides were stained with hematoxylin and eosin (H&E). For a better appreciation of the degree of hemosiderosis, a specific iron stain (Prussian blue stain according to Perls) was prepared for formol-fixed sections of the liver and spleen.

Statistical analysis. Body weights, hematology, and clinical pathology data were compared using either (1) the Kruskal-Wallis test and the adjusted U-test or adjusted Welsh-test as the post hoc test or (2) one-way analysis of variance (ANOVA) and the Dunnett test as the post hoc tests for nonparametric and parametric analyses, respectively (SAS 6.12-routines). Specialized endpoints (GPx, ferritin, iron) were analyzed with one-way ANOVA and the Tukey-Kramer post hoc test (BCTIC). Histopathological findings were compared with the concurrent control using Fisher's exact test as described by Gad and Weil (1989)Go. The proportion of erythrocytes with Heinz bodies was transformed prior to ANOVA analysis using the arcsine square root function. This is appropriate for percentages and proportions, because the transformed data more closely approximate a normal distribution than the non-transformed proportions (Sokal and Rohlf 1969Go). Transformed data were analyzed separately for normality of distribution. For all tests, the criterion for statistical significance was set at p < 0.05. Asterisks in figures and tables denote statistically significant differences to the concurrent air control group as follows: *p < 0.05 and **p < 0.01.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure Concentrations of Aniline
The mean (± SD) actual exposure concentrations were 9.2 ± 0.67, 32.4 ± 1.42, 96.5 ± 4.61, and 274.9 ± 8.54 mg/m3 (exposure for a maximum of 10 days, 3 determinations/day and chamber) and matched the targeted concentrations of 10, 30, 90, and 270 mg/m3. The actual breathing zone concentrations were in the range of 91%–98% of the nominally evaporated concentrations of aniline. The exact exposure regimen is included in Figure 1.



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FIG. 1. Determination of body weights of male rats exposed for 2 consecutive wks (6 hr/day, 5 days/wk) to aniline (mean ± SD). Vertical lines represent time points where 5 rats/group were sacrificed after exposure (days 0, 4, and 11) or during the post-exposure observation period (days 14 and 28).

 
Animal Observations, Body Weights, and Gross Necropsy
All animals survived the duration of study. Concentrations up to and including 30 mg/m3 were tolerated without clinical signs. Rats exposed to 90 mg/m3 and above were cyanotic (blue discoloration of the skin in areas easily observed), whereas those exposed to 270 mg/m3 also displayed tachypnea, labored breathing patterns, increased salivation, and an ungroomed hair-coat. All signs disappeared toward the following day and did not show any exacerbation during the course of the study. Rectal temperatures measured shortly after cessation of exposure on days 0, 4, and 11 were at all time points indistinguishable among the groups (range of means in the control group: 37.3°–38.6°C, aniline exposure groups: 36.6°–38.4°C). As depicted in Figure 1, body weights were not statistically significantly affected in any group during the course of the study. Gross necropsy findings of rats exposed to 90 mg/m3 and above provided evidence of dark discolorations and enlargement of spleens at all sacrifices beyond the first exposure week. Some discoloration of lungs was found; however, no consistent time-dependence or concentration-dependence existed. An increased incidence of discoloration of the thymus, including involution, and of the testes occurred at 270 mg/m3 (days 11 and 14).

Effect of Aniline Exposure on Key Blood Parameters
Hematological parameters were significantly affected at 90 and 270 mg/m3 starting on day 4, with maximum effects on days 11 and 14. They were characterized by a decrease in Hb, red blood cell count, and HCT, and an increase in reticulocyte counts and erythrocytes containing Heinz bodies (Table 1). The reticulocyte counts observed in controls on day 0 were twice as high as those observed at the subsequent time points. This change might be attributable to the immobilization stress caused by restraint of non-acclimatized rats to exposure tubes and the associated increased discharge of mature reticulocytes from the bone marrow. Rats exposed to 270 mg/m3 showed a significantly increased mean corpuscular hemoglobin and mean corpuscular volume (MCH and MCV), and the red blood cells in some rats (day 11 only) exhibited hypochromia and anisocytosis. With regard to the time-course, the hematological changes were maximal on the last exposure day (day 11) and showed some decrease in magnitude on the third post-exposure day (day 14); then, with the exception of the RBC, MCH, and MCHC, hematological changes subsided to the levels of the control group after the 2 week post-exposure. Thrombocytes were mildly, although significantly increased at the end of the 2-week exposure period (data not shown). Conclusive changes in the total leukocyte counts and leukocyte differentials did not occur at any time point (data not shown). The blood MetHb concentrations are summarized in Figure 2. In the blood sampled and determined immediately after cessation of exposure, MetHb was concentration-dependent, and then was significantly increased at 90 mg/m3 (days 4 and 11) and at 270 mg/m3 (days 0, 4, 11). The MetHb concentrations from blood samples taken in the post-exposure period were indistinguishable from the control. The differences in baseline MetHb concentrations observed during the exposure and post-exposure periods were due to the different methodologies used for determinations.


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TABLE 1 Hematology of Male Rats Exposed to Aniline up to 2 Weeks (6 h/day, 5 days/week), Followed by a Post-Exposure Period of 2 Weeks

 


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FIG. 2. Determination of the percentage of methemoglobin (MetHb) of male rats exposed for 2 weeks (6 h/day, 5 days/week) to aniline (mean ± SD, n = 5). For determinations during the exposure period, a hemoximeter method was used, and for the post-exposure period, the Evelyn and Malloy method was used. Asterisks denote statistically significant differences to the concurrent air control group: *p < 0.05; **p < 0.01.

 
Effect of Aniline Exposure on Key Clinical Pathology Parameters
The clinical pathology data considered focused on potential hepatotoxicity and erythrocyte (hemoglobin) catabolism by the end of the exposure and post-exposure periods and are summarized in Table 2. Statistical comparisons did not reveal any consistent concentration–response relationship considered to be of pathodiagnostic relevance except slightly altered bilirubin serum concentrations at 90 and 270 mg/m3. Bilirubin was increased or decreased by the end of the exposure and post-exposure periods, respectively. Some electrolytes (calcium, magnesium) were statistically significantly decreased at 30 mg/m3 and above, especially at the end of the post-exposure period (Table 2). Because of the lack of a clear dose-dependence from 30 to 270 mg/m3—in comparison to the control and 10 mg/m3 groups—these changes are likely not biologically significant findings. They appear to be secondary to changes in the concentrations of negatively charged counterions (anion gap), such as plasma proteins or bicarbonate, rather than reflecting any specific, aniline-induced disturbance in electrolyte homeostasis.


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TABLE 2 Clinical Pathology of Male Rats Exposed to Aniline up to 2 Weeks (6 h/day, 5 days/week), Followed by a Post-Exposure Period of 2 Weeks

 
Splenic Lipid Peroxidation and Total Iron Content of Spleen and Liver
The concentration-dependent and time-dependent changes of iron in homogenates of the spleen and liver are illustrated in Figures 3 and 4. Because of the noticeable changes in spleen weights during the course of the study (see below), the total splenic iron content was analyzed relative to both gram spleen weight (Fig. 3) and total organ weight (Fig. 4). Remarkable concentration-dependent and time-dependent increases in the total splenic iron content were noted in rats exposed to 90 and 270 mg/m3, when compared to the controls. As illustrated in Figure 4, the increase in total iron and splenomegaly are related. In contrast, the maximum iron content in liver tissue homogenates was transient, and a slight increase was observed at 270 mg/m3 only (Fig. 3). Based on a gram tissue level comparison, the maximum accumulation of iron in the liver and spleen exceeded the control levels by {approx}60% and {approx}500%, respectively.



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FIG. 3. Liver iron content (upper panel) and splenic (lower panel) iron content per gram organ weight of control and aniline-exposed rats. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. Values are means ± SD of five rats in each group. Asterisks denote statistically significant differences to the respective controls: *p < 0.05; **p < 0.01.

 


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FIG. 4. Splenic iron content per organ of control and aniline-exposed rats. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. Values are means ± SD of five rats/group and sacrifice. Asterisks denote statistically significant differences to the respective controls: *p < 0.05; **p < 0.01.

 
The degree of lipid peroxidation is shown in Figure 5. Lipid peroxidation, measured as the sum of malondialdehyde and 4-hydroxy-2(E)-nonenal, was significantly increased at 90 and 270 mg/m3. Despite the marked increase in lipid peroxidation from exposure day 0 to exposure day 4, an appreciable time-dependent progression between the later exposure days was not apparent. Changes subsided toward the level of the control group at the end of the post-exposure period, i.e., the aniline exposure groups were indistinguishable from the control.



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FIG. 5. Lipid peroxidation in the spleen homogenates of aniline-exposed rats. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. Values are means ± SD of five rats/group and sacrifice. MDA: malondialdehyde, HNE: 4-hydroxy-2(E)-nonenal. Asterisks denote statistically significant differences to the respective controls: *p < 0.05; **p < 0.01.

 
The correlation of the individual animals' total organ splenic lipid peroxidation and iron is shown in Figure 6. The comparison demonstrates that, during the exposure period, the increase of splenic lipid peroxidation and total iron was highly correlated (r2 = 0.93), whereas this high degree of correlation ceased to exist at the end of the post-exposure period. The determination of ferritin and GPx in the tissue homogenates of the spleen did not attain statistical significance, when compared with the respective controls (Fig. 7). However, despite the lack of any consistent concentration-dependence or time-dependence, GPx activity showed a tendency toward increase at the high exposure level.



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FIG. 6. Correlation of lipid peroxidation (LPO) in total iron in the spleen homogenates of aniline-exposed rats. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. Data points represent individual rats (five rats/group and sacrifice). Hexagons represent data obtained at the end of the post-exposure period (day 28).

 


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FIG. 7. Glutathione peroxidase (upper panel) and ferritin (lower panel) in the spleen homogenates of aniline-exposed rats. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. Values are means ± SD of five rats/group and sacrifice. Data are expressed relative to the respective controls.

 
Splenic and Liver Morphology and Organ Weights
Organ weight changes were restricted to significantly increased spleen weights (Table 3) occurring at 90 and 270 mg/m3. The weights of liver (Table 3), brain, lung, testes, heart, kidneys, and thymus did not show treatment-related effects (data not shown). Microscopic examination of the rats sacrificed on day 15 revealed changes in the spleen at 30 mg/m3 and above and in the liver at 270 mg/m3 (Table 4 and Fig. 8). The most striking morphological changes were observed in the spleen at 90 and 270 mg/m3 and included vascular congestion and splenic hemosiderin pigmentation. A concentration-dependent increased iron deposition was also observed (iron-positive stains). Extramedullary hematopoiesis in the spleen was observed at 30 mg/m3. Lesions indicative of specific splenic tissue damage were not observed. Liver sections stained with the iron-positive stain showed increased hemosiderosis, which gained statistical significance in the 270 mg/m3 exposure group. Bone marrow changes indicative of increased medullary hematopoiesis were seen in the histological sections of femur in rats exposed to 270 mg/m3 (evidenced by a decrease in marrow adipocytes). Spontaneous findings in the remaining tissues examined, including the lung, did not show any relation to exposure to aniline.


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TABLE 3 Liver and Spleen Weight and Organ/Body Weight Ratios of Male Rats Exposed to Aniline up to 2 Weeks (6 h/day, 5 days/week), Followed by a Post-Exposure Period of 2 Weeks

 

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TABLE 4 Incidence of Exposure-Related Histopathologic Lesions in Male Rats Exposed to Aniline up to 2 Weeks (6 h/day, 5 days/week), Followed by a Post-Exposure Period of 2 Weeks

 


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FIG. 8. Microscopic examination of splenic hemosiderin pigmentation, iron-positive stains, and extramedullary hematopoiesis of rats exposed for 2 weeks (6 h/day, 5 days/week) to aniline at 4 days after exposure termination. Cumulative incidence of graded findings in 5 rats/group. Sections were stained with H&E or Prussian blue.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Rats exposed to 90 and 270 mg aniline/m3 displayed a concentration-dependent, transient cyanotic appearance. Methemoglobinemia was the primary toxic response, occurring at 90 mg/m3 and above. The hypothetical, idealized time-course of the concentration-dependent MetHb formation during one exposure period is depicted in Figure 9, from which it can be deduced that the MetHb concentration observed at 30 mg/m3 was indistinguishable from that of the control group, based on hemoximeter determinations. The increased MetHb present at 90 mg/m3 after the fifth and later exposures, but not after the first exposure, may be associated with a decreased reduction capacity of erythrocytes as a result of recurrent MetHb-formation (Fig. 2) and ensuing oxidative stress. Similar observations have been made by Kim and Carlson, 1986Go). Grossman, et al. (1992)Go have shown that N-hydroxylated dapsone caused a rapid decrease in glutathione in rat erythrocytes, which limits the capacity of the RBC to reduce MetHb.



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FIG. 9. Calculated steady-state of MetHb formation and recovery using first-order kinetics. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. The first and last exposure days were days 0 and 11, respectively. Values are means ± SD of five rats/group and sacrifice.

 
For aniline, a substance that must be bioactivated by hepatic metabolism to become a MetHb-forming agent (Harrison and Jollow, 1987Go; Khan et al. 1998Go; Kiese, 1974Go), the magnitude of the formation of MetHb is highly contingent on both the route and the regimen used for administration or exposure. In dogs, a model that allows repetitive blood sampling without concomitant stimulation of hematopoiesis, it was shown that equal doses (15 mg/kg body weight) administered either by a single 4-h head-only inhalation exposure or by gavage, produced a {approx}5-times higher maximal MetHb concentration by gavage as by inhalation (Pauluhn, 2002Go). The exposure dose of 30 mg/m3 per 6 h/day used in this study is equivalent to approximately 12 mg aniline/(kg x bw day; for details see below). In contrast to dogs, no evidence of increased MetHb formation existed at this exposure level in rats (Figs. 2 and 9). Dogs that received the same dose by head-only exposure exhibited 5% MetHb after a 4-h exposure to 155 mg aniline/m3 (Pauluhn, 2002Go; MetHb value interpolated from Figure 6). Although other explanations might be plausible, this higher MetHb-forming capacity of dogs may be seen in context with the absence of any N-acetyltransferase activity in this species, whereas rats are known to be strong acetylators. Brodie and Axelrod (1948)Go demonstrated that the acetanilid is much less potent than aniline for MetHb-formation in vivo. These considerations support the general viewpoint, namely, that a meaningful and reliable extrapolation across different routes, regimens, dose ranges, and species requires an in-depth analysis of the magnitude and time course of the biologically active intermediary metabolites, especially phenylhydroxylamine. Therefore, for a toxicological assessment of exposure regimens of aniline similar to levels occurring in the workplace, the results from inhalation studies appear to be superior to results from studies using any other route of administration.

A compensated anemia was produced, as evidenced by an increase in the reticulocyte count and a decrease in hemoglobin concentrations, HCT, and red blood cell counts. The primary adverse effect appears to be governed by the potency of aniline-derived metabolites to cause methemoglobinemia and associated dysfunctions of erythrocytes. The other abnormalities observed could be explained as secondary to this type of hematotoxicity and included subsequent increases in erythrocyte damage and turnover, including anemia, RBC morphological alterations (e.g., Heinz bodies), and effects on the spleen (splenomegaly, hemosiderin accumulation, and increased hematopoietic cell proliferation), which gained significance at 90 and 270 mg/m3. The conspicuous increase in spleen weights, including the marked reticulocytosis of peripheral blood, returned to normal within the 2-week post-exposure period. Because there were no direct or primary microscopic abnormalities observed in the spleen by the end of the exposure period, the splenic effects appeared to be secondary to erythrocyte dysfunction.

At the high exposure level, the anemia was macrocytic, as indicated by an increase in MCV; at the end of the 2-week exposure period, it was somewhat hypochromic to normochromic, as indicated by a slight decrease in MCH concentration. In contrast, at the end of the 2-week post-exposure period the cells were macrocytic and hyperchromic. In regenerative anemias, hypochromasia is an expected finding in association with a reticulocytosis (reticulocytes are large, hypochromic cells). The slight hyperchromasia noted at the end of the post-exposure period is also typically seen in rats with slight decreases in reticulocyte counts. This decrease results from a population of relatively smaller cells with a normal complement of hemoglobin, and thus normal or increased MCHC. Despite the remarkable effects observed shortly after the 2-week exposure period, only minimal residual changes still existed in the high-concentration exposure group at the end of the post-exposure period. The presence of Heinz bodies is consistent with aniline-mediated, direct oxidative damage to the red blood cells, possibly through depletion of cellular reduction equivalents and antioxidants (Harvey, 1989Go). Heinz bodies appear only in the presence of unstable types of hemoglobin and are related to changes in membrane deformability and rigidity, but they do not induce mechanical membrane damage by themselves (Vácha, 1983Go). With regard to erythrocytotoxicity, 30 mg/m3 of alanine was tolerated without an adverse effect at any time point. The life-span of white blood cells and platelets remained unchanged under these conditions.

In mammals most heme is utilized for the oxygen-transport protein hemoglobin. The heme biosynthesis and degradation pathways affect cellular oxidant metabolism because both are closely linked with iron cycling. Free, i.e., non-chelated iron catalyzes autoxidations by Fenton and Haber-Weiss reactions, resulting in the production of reactive oxygen species (Bacon and Britton, 1990Go; Winterbourn, 1995Go). In fact, Ciccoli et al. (1999)Go have shown that hydroxylated metabolites of aniline induce iron release in erythrocytes in which marked formation of MetHb occurs. Under certain conditions, heme biosynthesis also generates reactive oxygen species (ROS). The rate-determining step in heme breakdown, the microsomal enzyme heme oxygenase (HO), has emerged as a central component of the mammalian stress response (Ryter and Tyrrell, 2000Go). HO may participate in a coupled cellular protection mechanism in which the iron storage protein ferritin ultimately provides protection by sequestering and oxidizing the iron released by the HO-catalyzed breakdown of heme. Red blood cell hemoglobin in intact, but senescing erythrocytes, undergoes degradation in the reticuloendothelial system of the liver, kidney, and especially the spleen, where HO activity is highest (Tenhunen et al., 1970Go). Free hemoglobin and heme released from MetHb may also enter the bloodstream during intravasal hemolysis (Garby and Noyes, 1959Go). In comparison to the nitro-substituted arylamines, aniline is among the least potent indirect inducers of MetHb (French et al., 1995Go). This supports the hypothesis that aniline-induced MetHb renders the erythrocytes relatively intact rather than causing frank intravascular hemolysis. The findings of this study suggest strongly that damaged cells are removed intact by the spleen and do not lyse into fragments in the general circulation, at least to any significant extent. Accordingly, for aniline, overwhelming predominance of uptake into the spleen, as distinct from the removal by the reticuloendothelial system in general, is evidenced by the relatively low liver uptake. This conclusion is not at variance from that of other authors observing similar findings (Jollow and McMillan, 2001Go). Under normal conditions, iron is transported between sites of absorption, storage, and utilization by transferrin, the only physiologically active chelate than can provide iron for the hemoglobin synthesis (Ponka, 1997Go). This process increases in complexity under conditions where massive amounts of iron prevail as a result of erythroclasia and heme catabolism and concomitantly increased reutilization at the same "site" because of the precipitously increased extramedullary splenic hematopoiesis. The absence of a microcytic, hypochromic anemia suggests that the iron from heme degradation is readily available for de novo heme synthesis. Hence, within the respective splenic compartments, iron is transported to intracellular sites of reutilization and/or storage in ferritin or hemosiderin, but this aspect of iron metabolism, including the elusive pool of "free", i.e., catalytically active iron and its cellular trafficking, remains enigmatic. Accordingly, the associated concentration-dependent and time-dependent increases in splenic iron, lipid peroxidation, and hypertrophy appear to be more likely linked to an excessively increased heme degradation and synthesis in the spleen than being causally linked to the mere presence of iron. Various studies have demonstrated the accumulation of "lipofuscin-like" pigments in erythrocytes exposed to oxidative stress. It was found that erythrocytes lost their deformability and were scavenged by spleen macrophages after peroxidation of their membrane lipids (Goldstein et al., 1980Go; Jain and Hochstein, 1980Go; Wilhelm and Herget, 1999Go). The comparison of the relative extent and time course of changes appears to corroborate the notion of this interrelationship (Fig. 10).



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FIG. 10. Comparison of relative extent of changes related to lipid peroxidation and iron in the homogenates of the spleen and spleen weights. Animals were exposed for 2 weeks (6 h/day, 5 days/week) to aniline. The first and last exposure days were days 0 and 11, respectively. Values are means ± SD of five rats/group and sacrifice. Data are expressed relative to the respective controls. Asterisks denote statistically significant differences to the respective controls: *p < 0.05; **p < 0.01.

 
A consistent time-dependent or concentration-dependent increase in the splenic iron-sequestering protein ferritin was not observed. At the end of the post-exposure period, the still moderately elevated levels of splenic iron were not associated with an appreciable increase in lipid peroxidation (Fig. 6), a time point at which the reticulocyte count in peripheral blood waned to the level of the control. Thus it is conceivable that the concentrations of endogenous membrane antioxidants in the spleen become increasingly depleted with increased concentrations of "free" heme, or that iron may render the spleen particularly susceptible both in terms of removal of damaged erythrocytes and associated oxidative stress at exposure levels causing frank methemoglobinemia. In contrast to the spleen, only a minute increase of total iron was observed in the liver.

The increased extramedullary hematopoietic cell proliferation in the spleen constitutes the most sensitive response, and it occurred in a concentration-dependent manner at 30 mg/m3 and above, whereas in the bone marrow an increased medullary hematopoietic response was observed only at 270 mg/m3. With regard to findings in the spleen, a significantly increased (hemosiderin) pigmentation and iron-positive staining (Prussian blue) was observed at 90 mg/m3 and above, with somewhat borderline effects at 30 mg/m3. As for iron in stores, it is difficult to know what this means. The stainable iron present in the form of hemosiderin is in a form that is almost certainly not readily available for any metabolic purpose (Cavill, 2002Go). Congestion of the spleen, corresponding to the splenomegaly detected at necropsy and increased spleen weights, were observed at 90 mg/m3 and above. In the liver, significantly increased (hemosiderin) pigmentation was restricted to the 270 mg/m3 exposure group. With regard to extramedullary splenic hematopoiesis, which serves only an accessory function, the findings from rats cannot be applied directly to humans. In the adult human the bone marrow plays the most important role as an erythropoietic organ, both under normal conditions and under conditions of hypoxia. In rats, however, the spleen satisfies an increased demand for erythrocytes in response to a hypoxic stimulus, whereas bone marrow and liver remain relatively inert (Pepelko, 1970Go; Ou et al., 1980Go), consistent with the findings of this study. This difference between human and rat can be explained anatomically by the larger capacity of human bone marrow, which is sufficient for the increased production of erythrocytes under hypoxic conditions; however, the rat, with its limited marrow space, has to rely on extramedullary erythropoiesis in the spleen (Seifert and Marks, 1985Go), and sometimes even in the liver (Crosby, 1983Go).

Thus in rats the spleen appears to be an important organ in the physiological response to compensate anoxia or an anemic state. In contrast, in human adults only certain pathological conditions accompanied by extramedullary hematopoiesis cause erythropoiesis localized mainly in the splenic sinuses (Rappaport, 1970Go). In rodents, the spleen's contribution to hematopoiesis is about 10% in normal animals; however, in particular for agents causing extensive hemolytic anemia, the splenic erythropoietic contribution can reach up to 80% of the total red cell production (Pantel et al., 1990Go). Moreover, a strong erythropoietic stimulus can induce five or six additional erythropoietic cell divisions in the spleen but only two or three additional divisions in the bone marrow. The difference between the erythropoietic sites in the splenic red pulp of human and rat may be explained by the fact that the spleen in small animals, such as rats, remains a normal hematopoietic organ through adulthood (Crosby, 1983Go), whereas in humans it ceases to have hematopoietic functions after birth (Freedman and Saunders, 1981Go; Stutte et al., 1986Go). Past investigations suggest that hematopoiesis in the fetal human spleen may be limited or even nonexistent (Ishikawa, 1985Go). In contrast to the proliferation of local erythroblasts in the rats' spleen, splenic hematopoiesis in humans is mediated by the immigration of erythroid cell precursors from the blood (Freedman and Saunders, 1981Go) and their proliferation in the splenic sinuses. The increased splenic erythropoiesis returns to normal mainly through erythrophagocytosis by macrophages after cessation of the stimulus, which is in line with the rapid recovery observed in this study.

Despite the uncertainties involved in the calculation of equivalent oral dosages, such conversion is attempted to make this study comparable to a chronic dietary study with aniline hydrochloride (CIIT, 1982Go). Based on a generic respiratory minute volume of rats in nose-only restrainers (1 l/min/kg-rat; Mauderly, 1986Go) the calculated exposure doses of this study were 3.3, 12, 35, and 99 mg aniline/(kg bw x day) for the actual exposure concentrations of 9.2, 32.4, 96.5, and 274.9 mg aniline/m3, respectively. In the 2-year feeding study in Fischer 344 rats the respective dietary dosages were 7, 22, and 73 mg aniline-HCl/(kg bw x day). Briefly, at the low dose, splenic hemosiderosis, increased splenic hematopoiesis, and mild reticulocytosis characterized major effects. At the intermediate dose, in addition to the findings observed at the low dose, evidence of MetHb formation and increased spleen weights existed. Heinz bodies, splenic inflammation, hyperplasia, fibrosis, and splenic tumors (sarcomas), including hepatic changes, were essentially restricted to the high-dose level. Most of the non-neoplastic splenic changes were observed after 12 months of study in the low-dose group and 6 months of study in the intermediate-dose group. Thus, notwithstanding the remarkable differences in dosing regimens and durations of studies, at 35–99 mg/(kg bw x day) inhalation exposure, a dose-dependent splenic toxicity related to increased oxidative stress, iron overloading, and exhaustion/destruction of the iron-sequestrating proteins is likely a cause of the effects occurring at 73 mg/(kg bw x day) after chronic dietary exposure. The calculated NOEL [3.3 mg aniline/(kg bw x day)] and borderline dose for increased splenic iron accumulation and increased lipid peroxidation [12 mg aniline/(kg bw x day)] observed after 2 weeks of inhalation exposure were also consistent with the findings of the chronic dietary study at 7 mg/(kg bw x day). This coincidence of findings supports the view that the etiopathologic mechanisms of splenic toxicity apparently reflect a recurrent acute erythrocytotoxicity that results from an extended MetHb formation. This, in turn, causes increased sequestration of (recurrently damaged and antioxygen depleted) erythrocytes in the spleen in a rat-specific manner, as well as liberation of redox active iron in this organ, which if excessive enough, is considered to be causally related to tumor formation in this organ.

To summarize, this repeated 2-week inhalation study on rats identified methemoglobinemia, the ensuing erythrotoxicity, and compensated anemia as the lead effects of aniline. With regard to erythrocytotoxicity and associated increased erythroclasia, 30 mg/m3 constitutes the NOAEC. A significantly increased compensatory splenic hematopoietic cell turnover occurred at 90 mg/m3 and above. Minimal focal responses slightly above the physiological level were observed by light microscopy at 30 mg/m3. Exposure to 10 mg/m3 was devoid of any significant effect. Therefore, the NOAEC is considered to be 30 mg/m3 because, in the circulating blood, there was no evidence of direct or indirect erythrocytotoxicity, increased erythroclastic effects, or adversity related to increased lipid peroxidation. With respect to the microscopic evidence of "minimally increased number of iron-positive and hematopoietic foci" in the spleen, the analytical quantitation of iron is considered to be superior to the microscopic resolution, applying conventional procedures. In this context, it is important to recall that extramedullary hematopoietic response of the spleen of rats has essentially no counterpart in humans. Therefore, because of the absence of any adverse erythrocytotoxic effects, the presence of a slightly increased compensatory extramedullary splenic hematopoiesis is considered to be a slightly raised homeostatic, small laboratory animal–specific response rather than an unequivocal biomarker of adversity. The NOAEC of 30 mg/m3 could be considered to be a safe level for workers by taking into account an adjustment factor of five (based on the findings from an inhalation study in dogs; Pauluhn, 2002Go) to accommodate for the polymorphism expressed by "poor acetylators" and the higher methemoglobin reductase activity in rats. This calculated value is consistent with the current workplace standard of 8 mg aniline/m3, based on MetHb and Heinz body formation in humans (DFG, 2003Go).


    ACKNOWLEDGMENTS
 
The author thanks B. Helmerstein, C. Pfeifer, and D. Zischka-Kuhbier for excellent technical assistance; Dr. M. Rosenbruch for histopathology data, I. Loof, G. Wasinska-Kempka, and U. Schmidt for hematology and clinical chemistry data; and A. Folkerts for the determination of aniline in exposure atmospheres.


    NOTES
 

1 For correspondence via fax: ++49 (202) 364589. E-mail: juergen.pauluhn{at}bayerhealthcare.com


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 Methods
 RESULTS
 DISCUSSION
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