James S. Bus

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.

The morphogenesis of testicular degeneration induced in rats by orally administered 2,5-hexanedione

The neurotoxic hexacarbon 2,5-hexanedione (2,5-HD) produces testicular atrophy in experimental animals. To examine the morphogenesis of the testicular lesion, 1.0% 2,5-HD was provided in the drinking water of adult F-344 rats for up to 6 weeks. After 3 weeks of administration, there were occasional large vacuoles in the basal region of the germinal epithelium. At 4 weeks, these vacuoles were much larger and more numerous; electron microscopy demonstrated that they were derived from the smooth endoplasmic reticulum. The vacuoles were preferentially associated with stages 12, 13, 14, and 1 of the spermatogenic cycle. Additionally, at 4 weeks there was a significant decrease in the number of tubules in stages 7 and 13, and a concomitant increase in the percentage of tubules in stages 3, 5, and 6. By Week 5, most Golgi-phase and cap-phase spermatids were visibly affected, showing margination of nuclear chromatin, and were becoming dissociated from Sertoli cells. Frequent multinucleated giant cells were seen and electron microscopy of these cells suggested that they were derived from fused spermatocytes or spermatids. After 6 weeks, fewer giant cells were present, most tubules contained cellular debris, and many showed empty lumina encircled by a thin ring of cytoplasm near the basement membrane. Interstitial tissue appeared unaffected. These studies indicate that the Sertoli cell is probably an initial target for 2,5-HD action in the testis.

A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites☆

Male Fischer-344 rats were given 100 μCi (14 mg/kg) [14C]catechol or [14C]hydroquinone by injection into the lateral tail vein. For a period of at least 24 hr, soluble radioactivity associated with either compound was retained in the bone marrow, but not in the liver or thymus. The amount of covalently bound radioactivity increased with time in all tissues examined and was significantly depressed in liver, white blood cells, and bone marrow in rats pretreated with Aroclor 1254, a regimen which protects against benzene toxicity. Potential enzymatic and nonenzymatic activation pathways for catechol, hydroquinone, and other known benzene metabolites were examined. In air-saturated 50 mm phosphate buffer (pH 7.4) at 37°C, only hydroquinone and 1,2,4-benzenetriol autoxidized. The oxidation product of hydroquinone had an uv absorption maximum (248 nm) identical to that of benzoquinone. With 250 units superoxide dismutase, hydroquinone autoxidation increased fivefold, whereas the oxidation of 1,2,4-benzenetriol was inhibited (4% of control). Epinephrine autoxidation, an indirect measure of superoxide anion generation, was stimulated by 1,2,4-benzenetriol and hydroquinone, but was barely detectable in the presence of catechol. Of the compounds studied, only benzoquinone augmented the oxidation of NADPH by a 3000g rat bone marrow supernatant. These data support a mechanism for benzene toxicity in which the formation of potentially cytotoxic metabolites, semiquinone, and quinone oxidation products and superoxide radicals, result from autoxidation of at least two polyphenol metabolites of benzene, hydroquinone, and 1,2,4-benzenetriol.

2,2,4-Trimethylpentane-induced nephrotoxicity: II. The reversible binding of a TMP metabolite to a renal protein fraction containing α2u-globulin

Trimethylpentane (TMP) produces nephrotoxicity in male but not in female rats. The toxicity is characterized by an increase in protein droplets in proximal convoluted tubular cells and an increase in the renal concentration of the male-rat-specific protein alpha 2u-globulin. Subcellular fractionation of the kidneys from male rats 24 hr after [3H]TMP administration showed that about 60% of the radiolabeled material was localized in the 116,000g supernatant. Column chromatography of this supernatant resolved the radioactivity into two components; one, which contained about 26% of the radiolabel, coeluted with alpha 2u-globulin and cross-reacted with an antibody specific for alpha 2u-globulin. The remaining component eluted in the low-molecular-weight range (less than 1000 Da) and was assumed to be TMP metabolites. Radiolabel from [3H]TMP in male rat urine also resolved into two components with about 0.1% of the radiolabel in urine coeluting with the alpha 2u-globulin-containing fraction. Radiolabel from TMP in male rat liver 116,000g supernatant and plasma and in female rat kidney 116,000g supernatant eluted as a single component in the low-molecular-weight range. Dialysis (1000-Da cutoff) of male kidney 116,000g supernatant led to a loss of the low-molecular-weight components, but nondialyzable radiolabel (about 20%) still coeluted with the alpha 2u-globulin after gel chromatography. Dialysis against 0.1% sodium dodecyl sulfate led to a loss of both the low- and high-molecular-weight radioactive material. These results suggested that the high-molecular-weight radioactive material was formed by the reversible binding of a radioactive component of TMP to a male-rat-specific protein. Gas chromatography-mass spectrometry of an ethyl acetate extract of the alpha 2u-globulin-containing fractions of TMP-treated male rat kidney 116,000g supernatant identified 2,4,4-trimethyl-2-pentanol as the only bound metabolite to alpha 2u-globulin. These studies provide the first evidence for a reversible binding between a metabolite of TMP and a male-rat-specific protein in the kidney and thus provide important insight delineating a potential mechanism of hydrocarbon-induced hyaline-droplet nephropathy.

2,2,4-Trimethylpentane-induced nephrotoxicity: I. Metabolic disposition of TMP in male and female Fischer 344 rats

2,2,4-Trimethylpentane (TMP), a component of unleaded gasoline, causes nephrotoxicity in male, but not in female, rats. In the present study, male and female Fischer 344 rats were treated with a single oral dose of [14C]TMP (4.4 mmol/kg; 2 microCi/mmol). Radiolabeled material in kidney, liver, and plasma was determined at 4, 8, 12, 24, and 48 hr after dosing. Maximum concentration of TMP-derived radioactivity in kidney, liver, and plasma of male rats was found after 12 hr (1252, 1000, and 403 nmol eq/g, respectively), whereas those measured in females were found after 8 hr (577, 1163, and 317 nmol eq/g, respectively). A selective retention of the TMP-derived radiolabel in the kidneys of male rats was noted when peak tissue concentration was expressed as a percentage of administered dose. Kidney concentrations of TMP-derived radiolabel increased in a nonlinear, but dose-dependent, manner; the kidney to plasma ratio was greater at low doses than at higher doses. Increased retention of radiolabel material in the kidney was associated with a significant increase in renal concentration of the male-rat-specific protein, alpha 2u-globulin, 24 and 48 hr after TMP administration. Total radioactivity collected in urine 48 hr after TMP administration was similar in males and females (32 and 31% of dose). Identification and quantitation of the urinary metabolites of TMP showed that both male and female rats metabolize TMP via the same pathway and at a similar rate. Female rats, however, excreted more conjugates of 2,4,4-trimethyl-2-pentanol in urine than males. 2,4,4-Trimethyl-2-pentanol was the major metabolite present in the male rat kidney, but was absent in the female rat kidney. The renal retention of 2,4,4-trimethyl-2-pentanol appears to account for the delayed clearance observed in the disposition of [14C]TMP-derived radiolabel. Based on the concomitant accumulations in renal alpha 2u-globulin concentration and renal 2,4,4-trimethyl-2-pentanol concentration, an association is speculated between these two components. The male-rat-specific accumulation of 2,4,4-trimethyl-2-pentanol may therefore reflect the accumulation of a metabolite-alpha 2u-globulin complex. This may be relevant to the male-rat-specific nephrotoxicity produced by TMP.

Studies of lesions induced in the testis and epididymis of F-344 rats by inhaled methyl chloride

Experiments were carried out in rats to characterize the development of the testicular and epididymal lesions and any associated effects on reproductive hormones. Adult F-344 rats were exposed to 3500 ppm methyl chloride (MeCl) 6 hr/day for 5 days, not exposed for 3 days, and exposed again for 4 days. The first consistent testicular lesion was a delay in spermiation which appeared on Day 9. Subsequently, germinal epithelial vacuolation and cellular exfoliation became widespread as exposure continued. All animals killed after 19 days also displayed bilateral epididymal granulomas in regions 5 or 6 of the cauda epididymis. The nature and distribution of inflammatory cells indicated that the primary neutrophilic response may be against the tubular epithelium and not extravasated sperm. After 5 days of exposure, circulating testosterone was below 6 ng/ml (control: 120 +/- 31 ng/ml). Both MeCl exposed and control animals responded similarly to challenge with hCG and LHRH ethylamide, suggesting that Leydig cell and gonadotrope function was unaffected. It is proposed that MeCl acts centrally to lower circulating testosterone. Nonprotein sulfhydryls were depleted in liver, testis, and epididymis after MeCl exposure, but not in whole blood. This finding indicates that sulfhydryl depletion is not due to direct alkylation, but is enzymatically mediated. Sulfhydryl depletion did not correlate with lesion development. It was concluded that the initial testicular effects of MeCl are directed at either the late stage spermatids or the Sertoli cells with a resultant delay in spermiation.

The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride

Rat liver microsomes metabolized methyl chloride to formaldehyde at a rate 15-fold less than the rate of benzamphetamine demethylation. The reaction rate was stimulated approximately 2-fold in microsomes from phenobarbital-pretreated rats and was inhibited by addition of SKF-525A, carbon monoxide, metyrapone, and hexobarbital to the microsomal suspension, indicating dependence on cytochrome P-450. The in vivo incorporation of 14CH3Cl into liver macromolecules, previously shown to reflect metabolism to CH3Cl to formate, was not significantly altered by SKF-525A, Aroclor 1254, or 3-methylcholanthrene pretreatment of rats, although pretreatment with phenobarbital produced a 35 and 28% increase in 14CH3Cl uptake into liver lipid and acid-insoluble material, respectively. Pretreatment with phenobarbital increased the in vivo metabolism of 14CH3Cl to 14CO2 (also derived from a formate intermediate) by 19%, but had no effect on urinary metabolites derived from 14CH3Cl. SKF-525A inhibited 14CO2 production from 14CH3Cl by 30% and also had no effect on urinary excretion of 14C. In contrast, pretreatment with diethylmaleate inhibited 14CH3Cl incorporation into liver macromolecules by 70 to 85%, and lowered 14CO2 expiration and urinary 14C excretion by 52 and 60%, respectively. S-Methylcysteine pretreatment produced a similar inhibition of 14CH3Cl incorporation and metabolism to 14CO2; urinary excretion of 14C, however, was approximately doubled. 14CH3Cl uptake into liver was also stimulated by cysteine pretreatment. These results indicate a strong dependence of CH3Cl metabolism on tissue nonprotein sulfhydryl content and suggest a possible role for cytochrome P-450 in the in vivo metabolism of CH3Cl. A scheme for the metabolism of CH3Cl is postulated which involves initial reaction with glutathione, and sequential metabolism of the conjugate to S-methylcysteine, methanethiol, and formaldehyde.

The effects of 2,5-hexanedione on reproductive hormones and testicular enzyme activities in the F-344 rat

Abstract The chronic administration of 2,5-hexanedione (2,5-HD) to experimental animals can cause azoospermia and morphologic changes in central nervous system (CNS) areas related to visual and motor function. The present experiments were designed to determine the degree of CNS involvement in the testicular lesions seen after 2,5-HD administration. Additionally, activity measurements were made of some enzymes found in specific cell types in the testes. The 2,5-HD was administered as a 1% solution in the drinking water to adult male F-344 rats. Treated rats, pair fed controls, and ad libitum controls were killed after 1, 3, and 6 weeks of 2,5-HD treatment. The circulating levels of testosterone and the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), were not depressed at any time measured. After 6 weeks, the testes were azoospermic; this coincided with a rise in LH and FSH. After 3 weeks of 2,5-HD treatment, when the testes were morphologically normal, testicular activity of the Sertoli-cell-specific enzymes, β-glucuronidase and γ-glutamyl transpeptidase, were decreased. The testicular enzyme profile after 6 weeks was similar to that seen in the azoospermic, cryptorchid testis. Activities of hepatic β-glucuronidase and acid phosphatase were decreased at all time points. The data indicate that 2,5-HD does not act via the central gonadotropin control systems to induce azoospermia, and that demonstrable changes in Sertoli cell biochemistry occur prior to visible morphologic changes in the testis.

Reproductive effects of inhaled methyl chloride in the male Fischer 344 rat: II. Spermatogonial toxicity and sperm quality

Studies were performed to assess the effects of inhaled methyl chloride (MeCl) on sperm quality and testicular histopathology in the Fischer 344 rat. Adult male F-344 rats were exposed to 1000 or 3000 ppm MeCl 6 hr/day for 5 days, or received an ip injection of 0.2 mg triethylenemelamine (TEM)/kg on the afternoon of Day 5. Five males from a control group and each of the three treatment groups were killed weekly for 8 weeks, and five more from the control and 3000 ppm groups at Week 16 postexposure. Male rats from the 1000 ppm MeCl- and TEM-exposed groups exhibited no consistent difference from control males in any parameter measured. In the 3000 ppm MeCl group, over 50% of the males had uni- or bilateral sperm granulomas in the cauda epididymis. Testes weights were significantly depressed in these males by 3 weeks postexposure, and remained depressed as late as 8 weeks after treatment. Testicular spermatid head counts were significantly lower by 2 weeks postexposure, and the testes showed a characteristic cytotoxic response to MeCl exposure, including a delay in spermiation, chromatin margination in round spermatids, epithelial vacuolation, luminal exfoliation of spermatogenic cells, and multinucleated giant cells. Counts of repopulating seminiferous tubule cross sections and of testicular sperm heads at 7 weeks postexposure indicated that 60 to 70% of the spermatogonial stem cells had been killed by MeCl exposure. Sperm isolated from the vasa deferentia had significantly depressed numbers and an elevated frequency of abnormal sperm head morphology by Week 1 postexposure, and significantly depressed sperm motility and an elevated frequency of headless tails by Week 3 postexposure. All parameters measured in the testes and in vasa deferentia sperm showed varying degrees of recovery, and all were near the normal range by Week 16 postexposure except sperm count. These data suggest that the previously reported high preimplantation loss caused by methyl chloride exposure may be primarily due to cytotoxic effects on the testis and epididymal epithelium, rather than to direct genotoxic effects on the sperm.

Reproductive effects of inhaled methyl chloride in the male Fischer 344 rat: I. Mating performance and dominant lethal assay

Methyl chloride (MeCl) is a direct-acting mutagen in bacteria, induces renal adenocarcinomas in male mice, and is a potent reproductive toxicant in the male Fischer 344 (F-344) rat. A dominant lethal assay was performed to examine the possibility that MeCl may be a germ cell mutagen in the F-344 male rat. Groups of 40 rats each were exposed to 0, 1000, or 3000 ppm MeCl 6 hr/day for 5 consecutive days, or received a single ip injection of 0.2 mg triethylenemelamine (TEM)/kg as a positive control. Each male was bred to a single female weekly for 8 weeks, and the standard criteria of dominant lethal tests were recorded. Exposure to 1000 ppm MeCl caused no consistent change in any parameter relative to control values. TEM caused increases in pre- and postimplantation loss indices in Weeks 1 to 5 postexposure, corresponding to sperm exposed in the epididymis and as early to late stage spermatids in the testis. Exposure to 3000 ppm MeCl caused a slight increase in postimplantation loss at Week 1 postexposure only, i.e., in sperm exposed in the epididymis or vas deferens, and increases in preimplantation loss throughout the 8 weeks postexposure. Fertility of the 3000 ppm MeCl-exposed males was significantly decreased by Week 2 after exposure, and never recovered to control values during the period of observation. It is suggested that the increased preimplantation losses are due, at least in part, to cytotoxic rather than genotoxic effects. High concentrations of inhaled MeCl, however, do induce dominant lethal mutations (reflected as an increased level of postimplantation fetal deaths) in sperm located within the vas deferens and epididymis at the time of exposure.