Douglas E. Rickert

Benzene disposition in the rat after exposure by inhalation

Abstract Little information is available on benzene disposition after exposure by inhalation despite the importance of this route in man. Benzene metabolites as a group have been measured in bone marrow, but quantitation of individual metabolites in this target tissue has not been reported. Male Fischer-344 rats were exposed to 500 ppm benzene in air and the uptake and elimination was followed in several tissues. Concentrations of free phenol, catechol, and hydroquinone in blood and bone marrow were also measured. Steady-state concentrations of benzene (11.5, 37.0, and 164.0 μg/g in blood, bone marrow, and fat, respectively) were achieved within 6 hr in all tissues studied. Benzene half-lives during the first 9 hr were similar in all tissues (0.8 hr). A plot of amount of benzene remaining to be excreted in the expired air was biphasic with t 1 2 values for the α and β phases of 0.7 and 13.1 hr, respectively. Phenol was the main metabolite in bone marrow at early times (peak concentration, 19.4 μg/g). Catechol and hydroquinone predominated later (peak concentrations, 13.0 and 70.4 μg/g, respectively). Concentrations of these two metabolites declined very slowly during the first 9 hr. These data indicate that free catechol and hydroquinone persist in bone marrow longer than benzene or free phenol.

Dinitrotoluene: Acute Toxicity, Oncogenicity, Genotoxicity, and Metabolism

Dinitrotoluene (DNT) is a major commodity chemical; over six hundred million pounds of DNT were used in the chemical industry in 1980. Interest in the toxicology of this important chemical was greatly increased when separate oncogenicity assays yielded the conflicting results that DNT was either not hepatocarcinogenic or produced a 100% incidence of hepatocellular carcinomas in male rats in one year. Research revealed pronounced differences in the activity of the DNT isomers and provided the reason for the dissimilar results of the various carcinogenicity studies. Cell culture genetic toxicology assays failed to predict the potent carcinogenic activity of any isomer of DNT. Only when the complex pattern of metabolic activation of DNT began to unfold and genotoxic activity was assessed in the appropriate target organ in the intact treated animal was the potent genotoxic activity of DNT revealed, and the reasons for the negative in vitro results understood. The DNTs have been extensively tested for reproductive effects in animals and humans, and the metabolism and disposition of each of the six possible isomers have been studied. This work has provided valuable information in establishing the risk of these compounds to humans.

Metabolism of Nitroaromatic Compounds

There appear to be two major pathways for the metabolism of nitrobenzene and substituted nitrobenzenes. The first of these is reduction of the nitro group to yield aniline or substituted anilines. For nitrobenzene and perhaps for pentachloronitrobenzene, reduction of the nitro group to the amine is accomplished by bacteria of the gastrointestinal tract. Addition of a second nitro group results in easier reduction of one of the nitro groups on dinitrobenzenes, since they can be reduced under aerobic conditions by hepatic and erythrocyte enzymes. Bacterial reduction of the dinitrobenzenes is probably not quantitatively important in vivo. The second pathway is replacement of a nitro group by glutathione. The relative importance of this pathway compared to nitro group reduction depends upon the compound. It has not been demonstrated to occur for nitrobenzene. It is the major route of metabolism for 1,2-dinitrobenzene but is not an important route for 1,3- or 1,4-dinitrobenzene in hepatocytes. Tetrachloronitrobenzene isomers in which the nitro group is flanked by chlorines and pentachloronitrobenzene undergo nitro group replacement, but 2,3,4,5-tetrachloronitrobenzene does not. The most important pathway for the metabolism of mononitrotoluenes is methyl group oxidation. All quantitatively important metabolites are apparently formed from the nitrobenzyl alcohols. The mono- and dinitrotoluenes are not significantly reduced to isolatable metabolites by mammalian enzymes in vivo; intestinal microflora reduce these compounds after biliary excretion of the nitrobenzyl glucuronides. The little available evidence suggests that this is not the case for trinitrotoluenes. Urinary metabolites retain the methyl group and bear one or two amino groups. This suggests either that mammalian enzymes are capable of reducing the nitro groups of trinitrotoluenes in vivo or that intestinal microflora gain access to unmodified trinitrotoluene. The nitropolycyclic aromatic hydrocarbons are apparently metabolized by both nitro reduction and ring oxidation. There is good evidence, at least for 1-nitropyrene and 6-nitrobenzo[a]pyrene, that nitro reduction occurs in intestinal microflora. the complexities of foreign compound metabolism are well illustrated by the biotransformation of the nitroaromatic compounds. Positional isomers are preferentially metabolized by markedly different pathways. Substitution to different degrees or with different functional groups greatly affects the types of metabolites formed. Yet these compounds also offer opportunities for understanding the mechanisms of foreign compound metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzene is metabolized and covalently bound in bone marrow in situ

In trod uc t ion Benzene is a well-known myelotoxic agent which has recently been implicated as a leukemogen [1]. The mechanism by which benzene induces myelotoxic i ty is not understood. Myelotoxici ty could result from the action of the parent compound or any of the well-known metabolites of benzene such as phenol, catechol, hydroquinone, 1,2,4-benzenetriol or trans, transmuconic acid [2--4]. Quantitatively, the liver is the major site of benzene metabolism in the body [ 5] ; however, the relative importance of metabolism in the liver with respect of myelotoxic action is unknown. The intermediate(s) responsible for bone marrow toxici ty may be synthesized in the liver and transported to marrow where they act directly; they may require additional metabolism in bone marrow to form the ultimate toxic intermediate; or, as an alternative, bone marrow metabolism of benzene may be the only prerequisite for benzene induced myelotoxici ty. The possibility that benzene can be metabolized directly in bone marrow, the principal target organ, has no t been explored [6] . Phenol, catechol and hydroquinone are found in rat blood and bone marrow following inhalation of benzene [ 7], but the sites of formation of these metaboli tes have no t been established, nor has the metabolic contr ibut ion of the bone marrow been ascertained. The principal objective of this work was to determine if bone marrow is capable of metabolizing benzene independent of metabolism in the liver.

A critical review of the literature on nitrobenzene toxicity

This literature review encompassing information available through 1980 and limited coverage in 1981, emphasizes results useful in assessing the potential toxic effects of nitrobenzene to man. Nitrobenzene exposure in man or experimental animals is most often associated with methemoglobinemia. Histopathologic changes also are observed in the hemato-lymphoreticular system, central nervous system, and liver. In addition, lesions have been reported in adrenals and testes. No information was found on carcinogenic or teratogenic potential, fertility, or reproductive effects of nitrobenzene. Results from Ames Salmonella assay are negative but test procedures are questionable; metabolites give positive results. Metabolism of nitrobenzene involves either oxidation or reduction yielding p-aminophenol and p-nitrophenol, and other reduced intermediates. From the foregoing, several aspects of nitrobenzene toxicity have been identified which warrant further study. Recommendations are made for chronic and subchronic exposure of test animals via inhalation to assess various toxicological endpoints. In addition, the relationship of nitrobenzene metabolism to its toxicity needs to be established. Genotoxic effects of nitrobenzene also need study.

Elimination and metabolism of permethrin isomers in rainbow trout

Abstract Although permethrin, a synthetic pyrethroid insecticide, is highly toxic to fish, its toxicity to mammals is comparatively low. The distribution and metabolism of the cis - and trans -permethrin isomers were studied in rainbow trout to evaluate the role of these parameters in the differential toxicity of permethrin to fish and mammals. Both [ 14 C]permethrin geometrical isomers were readily taken up and eliminated by rainbow trout. Elimination half-lives for [ 14 C]permethrin residues in trout tissues, with the exception of fat, were in the magnitude of hours. High concentrations of a polar metabolite were found in bile within 4 hr of cis - and trans -permethrin exposure. Analysis by β-glucuronidase treatment, analytical thin-layer chromatography, and gas chromatography-mass spectroscopy indicated that the metabolite was the glucuronide conjugate of 4′-HO-permethrin. Urine contained a small amount of a polar metabolite that was resistant to hydrolysis by β-glucuronidase but was cleaved to some extent by aryl sulfatase. The relative absence of permethrin hydrolysis products in trout bile and the small amount of radioactivity excreted in urine suggested that the ability of rainbow trout to hydrolyze permethrin, in vivo , was minimal.

Physiologically based pharmacokinetic model for methanol in rats, monkeys, and humans.

The pharmacokinetics of methanol and formate were characterized in male Fischer-344 rats and rhesus monkeys exposed to methanol vapor concentrations between 50 and 2000 ppm for 6 hr. End-of-exposure blood methanol concentrations were not directly proportional to the atmospheric concentration. The methanol exposures did not cause an elevation in blood formate concentrations. After an intravenous dose of [14C]methanol in rats, metabolism, exhalation, and renal excretion contributed 96.6, 2.6, and 0.8%, respectively, to the elimination of blood methanol concentrations. These values and the calculated renal methanol extraction efficiency (0.007) are nearly identical to those for humans after low doses of methanol. A physiologically based pharmacokinetic model was developed to simulate the in vivo data. In order to simulate the observed blood methanol concentrations in the inhalation studies in rats, a double pathway for methanol metabolism to formaldehyde was used. One path used rodent catalase Km and Vmax values and the other used a smaller Km and Vmax to simulate an enzyme with a higher affinity and lower capacity. The lack of proportionality observed in end-of-exposure blood methanol concentrations may be due to saturation of an enzyme with higher affinity and lower capacity than catalase. The physiologically based pharmacokinetic model was modified to simulate the monkey data and was scaled-up for humans. In order to simulate the monkey blood methanol concentrations, the use of rodent catalase parameters for methanol metabolism was required. This finding suggests that primates and rodents may be similar in the initial step of methanol metabolism after low methanol doses. Previously published human urinary methanol excretion data was successfully simulated by the model. The models were used to predict the atmospheric methanol concentration range over which the laboratory species exhibit quantitative similarities with humans. Below 1200 ppm, all three species exhibit similar end-of-exposure blood methanol concentrations and a linear relationship between atmospheric and blood methanol concentrations. At higher atmospheric concentrations, external and internal methanol concentrations increase desparately, suggesting that delivered dose rather than exposure concentration should be used in interpreting data from high-dose studies.

Metabolism and excretion of 2,4-[14C]Dinitrotoluene in conventional and axenic Fischer-344 rats.

Comparisons of in vitro reduction of 2,4-dinitrotoluene (2,4-DNT) by cecal microflora and liver have indicated that microflora may play a large role in the in vivo metabolism of 2,4-DNT to reduced metabolites. Furthermore, reduction of 2,4-DNT by cecal microflora produces nitroso and, presumably, hydroxylamino intermediates which may account for the toxic actions of 2,4-DNT, including hepatocarcinogenesis. This study examines the metabolism, excretion, and hepatic covalent binding of 2,4-DNT in conventional, DNT-fed, and axenic Fischer-344 rats in order to define more precisely the role of DNT pretreatment and intestinal microflora in the disposition and toxicity of 2,4-DNT. No differences in 2,4-DNT disposition were produced by 30 days of feeding DNT (35 mg/kg/day) in the diet of male or female rats. Axenic males and females excreted less of a dose of 2,4-DNT in the urine than did conventional animals, and half-times for excretion of 4-(N-acetyl)amino-2-nitrobenzoic acid (4NAC2NBA), 2,4-dinitrobenzoic acid, 2-amino-4-nitrobenzoic acid (2A4NBA), and 2,4-dinitrobenzyl alcohol glucuronide were longer in axenic males than in conventional males. In axenic females half-times for excretion of only 4NAC2NBA and 2A4NBA were longer than in conventional females. Amounts of 4NAC2NBA and 2A4NBA excreted by axenic animals were 1/10th to 1/5th those excreted by conventional animals. Hepatic covalent binding was decreased by half in axenic animals. These data suggest that intestinal microflora play a major role in the appearance of reduced urinary metabolites and of covalently bound material after 2,4-DNT administration.

Induction of hepatic and testicular lesions in fischer-344 rats by single oral doses of nitrobenzene

Since the acute toxicity of nitrobenzene (NB) in rats has not been characterized, experiments were performed to ascertain the possible deleterious effects of NB in different tissues of the male Fischer-344 rat. Rats were given single oral doses of NB (50-450 mg/kg) and at the time of sacrifice, 25 tissues were removed and examined histologically by light microscopy. Histopathological changes induced by a single oral dose of NB consistently involved only the liver and testes. One rat receiving 450 mg NB/kg had a microscopic cerebellar lesion. Hepatic centrolobular necrosis appeared inconsistently in rats given various doses of NB, while hepatocellular nucleolar enlargement was consistently detected in rats given doses of NB as low as 110 mg/kg. These data suggest that nucleolar enlargement was independent of cell death and subsequent regeneration. Testicular lesions were confined to the seminiferous tubules and consisted of necrosis of the primary and secondary spermatocytes with the appearance of multinucleated giant cells between one and four days after administration of NB 300 mg/kg. Necrotic debris and decreased numbers of spermatozoa were seen in the epididymis as early as three days after NB administration. The NB-induced methemoglobinemia does not appear to be solely responsible for the formation of early lesions in the rat liver, testes, or brain, since sodium nitrite administration, at dosages which produced methemoglobinemia equivalent to that of NB, did not produce any histopathological changes. Thus, the observed liver and testicular damage are probably due to a direct effect of NB or its metabolites.

Metabolism of 2,4-dinitrotoluene by intestinal microorganisms from rat, mouse, and man.

Abstract Dinitrotoluene (DNT) is an industrial chemical of importance in the production of urethane foams and elastomers. The technical grade material is a hepatocarcinogen in rodents but shows sex- and species-dependent differences in potency. We have studied the pathways of metabolism of 2,4-dinitrotoluene (2,4-DNT), the major component of technical material, in the cecal microflora of male rats, female rats, male mice, and in human feces and ileal contents. 2,4-DNT was not metabolized by any of these preparations in the presence of oxygen. Under anaerobic conditions an ordered sequence of reductive metabolism was observed. The 2- and 4-nitro groups were reduced to amino groups via nitroso intermediates which were identified by GC-MS. The reduction of the nitroso intermediate to the amino compound is presumed to involve a hydroxylamino intermediate which could not be isolated. The aminonitro compounds were then reduced to diaminotoluene. No intermediates in this sequence could be isolated. No sex- or species-dependent differences in the pathways of metabolism were observed and only small species-dependent differences in the rate of metabolism of 2,4-DNT were observed. It is concluded that the intestinal microflora of rodents represent the major site of reductive metabolism of 2,4-DNT and may play an important role in the carcinogenic action of DNT isomers.