Institute of Toxicology, Bayer HealthCare AG, D-42096 Wuppertal, Germany
Received March 8, 2004; accepted June 1, 2004
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ABSTRACT |
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Key Words: methemoglobin; bone marrow; spleen; liver; hematotoxicity; aniline; lipid peroxidation; iron; oxidative stress; inhalation.
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INTRODUCTION |
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In spite of its structural simplicity, the metabolism of aniline is complex (Grossman and Jollow, 1986; Lenk and Sterzl, 1982
). 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, 1987
; Ferrali et al., 1997
; Grossman and Jollow, 1986
; Harrison and Jollow, 1987
; Jenkins et al., 1972
; Jensen and Jollow, 1991
; Khan et al., 1995a
, 1995b
, 1997
, 1998
, 1999a
, 1999b
, 2000
, 2003a
, 2003b
; Kiese, 1974
). 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, 2002
). The difference in potency can be attributed to differences in the extent of bioactivation or inactivation of reactive intermediates. As detailed by Kiese (1974)
and Akintowa (2000)
, 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, 2002
; Pauluhn and Mohr, 2001
). Previous work by Kim and Carson (1986)
has shown that following inhalation exposure of rats to aniline the half-life of MetHb was as short as 75 min (
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, 2001
), 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, 1983
) and EU animal welfare regulations (Council of the European Communities, 1986
).
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Methods |
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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., 2002), 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 23 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
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 011), 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, 2001). 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 analogse.g., Hejtmancik et al. (2002)
, Khan et al. (1995a
,b
, 1997
, 1998
, 1999a
,b
, 2000
; 2003a)
, and Nair et al. (1986)
.
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: 2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level:
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, 1994
). 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), -glutamyltranspeptidase (
-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, 2001
), 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, 1978
) using Triton X-100 as lysing agent according to Anders and Chung (1984)
, 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)
. 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 4080 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 ferritinHRP 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). 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 1969
). 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.
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RESULTS |
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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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DISCUSSION |
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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, 1989). 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, 1983
). 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, 1990; Winterbourn, 1995
). In fact, Ciccoli et al. (1999)
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, 2000
). 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., 1970
). Free hemoglobin and heme released from MetHb may also enter the bloodstream during intravasal hemolysis (Garby and Noyes, 1959
). In comparison to the nitro-substituted arylamines, aniline is among the least potent indirect inducers of MetHb (French et al., 1995
). 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, 2001
). 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, 1997
). 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., 1980
; Jain and Hochstein, 1980
; Wilhelm and Herget, 1999
). 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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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, 2002). 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, 1970
; Ou et al., 1980
), 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, 1985
), and sometimes even in the liver (Crosby, 1983
).
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, 1970). 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., 1990
). 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, 1983
), whereas in humans it ceases to have hematopoietic functions after birth (Freedman and Saunders, 1981
; Stutte et al., 1986
). Past investigations suggest that hematopoiesis in the fetal human spleen may be limited or even nonexistent (Ishikawa, 1985
). 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, 1981
) 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, 1982). Based on a generic respiratory minute volume of rats in nose-only restrainers (1 l/min/kg-rat; Mauderly, 1986
) 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 3599 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 animalspecific 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, 2002) 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, 2003
).
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ACKNOWLEDGMENTS |
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NOTES |
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1 For correspondence via fax: ++49 (202) 364589. E-mail: juergen.pauluhn{at}bayerhealthcare.com
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