Circadian variation in lipid peroxidation induced by benzene in rats

Time-dependent effect of benzene, a potent carcinogenic industrial solvent, on lipid peroxidaiton and associated mechanisms has been studied in liver and kidney of rats. Significant differences were observed in the values of urinary phenol, microsomal malondialdehyde, reduced glutathione (GSH) and cytochrome P4502E1 in rats treated with benzene in morning and evening hours. Higher were the values for urinary phenol and hepatic microsomal malondialdehyde in rats administered benzene in evening hours. Contrarily, higher were the values for GSH and cytochrome P4502E1 in rats treated with benzene in morning hours. Increased microsomal lipid peroxidation has been attributed to low GSH status, whereas increased phenol concentration could be related to low activity of cytochrome P4502E1 in the liver of rats in evening hours. It is concluded that circadian rhythmicity in hepatic drug metabolizing enzyme system and GSH contributes in toxicity of benzene. The results are important from occupational health point of view.     

Metabolism of benzene and phenol in macrophages in vitro and the inhibition of RNA synthesis by benzene metabolites

Benzene may affect hemopoiesis by damaging the bone marrow stroma that provides the microenvironment for hemopoiesis. A possible target of benzene toxicity in the stroma is the macrophage, which is a major source of protein factors required for the proliferation and differentiation of progenitor cells. As an initial approach towards understanding whether benzene inhibits hemopoietic factor production in bone marrow stroma, the metabolism of benzene and phenol has been studied and the effect of benzene and its metabolites on macrophage RNA synthesis has been examined. Benzene is not metabolized in macrophages but phenol, the major metabolite of benzene in bone marrow, is converted by peroxidase in the macrophage to both free metabolites and species which covalently bind to cellular macromolecules. Benzene and its metabolites inhibited RNA synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 5 × 10^–3M for benzene, 2.5 × 10^–3 M for phenol, 2.5 × 10^–5 M for hydroquinone, and 6 × 10^–6 M for p-benzoquinone; this inhibition was not attributable to loss of cell viability. Benzene, possibly by an inhibition of uridine transport into macrophages, and phenol, by its conversion to covalently binding species, inhibit RNA synthesis in macrophages and thus may inhibit the synthesis of colony stimulating factors required for hemopoiesis.Abbreviations CFU-G / M colony forming unit-granulocyte / macrophage - FCS fetal calf serum - IC₅₀ molar concentration causing 50% inhibition - PBS phosphate buffered saline     

Interactive inhibition of erythroid 59Fe utilization by benzene metabolites in female mice

Using radioiron uptake into erythrocytes as a measure of hematopoiesis, it was demonstrated that benzene inhibited bone marrow function in female mice. Hydroquinone was marginally effective, but the inhibition occurred only at the highest dose tested (100 mg/kg). The combination of phenol and hydroquinone was more effective in reducing erythrocyte production than either chemical given alone. Catechol given alone was not inhibitory but when phenol was added to catechol, erythropoiesis was suppressed, as observed for the phenol and hydroquinone combination. It appears that benzene toxicity may be the result of cooperative inhibitory effects produced by its metabolites.     

Lack of in vivo DNA binding of mercaptobenzothiazole to selected tissues of the rat

In this study, the in vivo binding of 14C-labelled 2-mercaptobenzothiazole (MBT) to DNA was investigated. Male and female Fischer 344 rats were gavaged with 375 mg MBT/kg body weight and killed 8 hours later. DNA was extracted from the liver, adrenal glands, pituitary gland, pancreas, and bone marrow and the amount of radioactivity associated with the DNA was determined. Results from this study indicate that MBT does not significantly bind to DNA from any of the tissues examined. CBI values for liver for the 3 methods of purification were -1-3 which are on the low end of the covalent binding index. The CBI values for the other tissues were always less than 1. Other chemicals with similar CBI values include estrone and diethylstilbesterol. Strong hepatocarcinogens such as dimethylnitrosamine and aflatoxin have CBI values ranging from 6000 to greater than 20000.     

Effects of ethanol and phenobarbital administration on the metabolism and toxicity of benzene

Effects of ethanol- and phenobarbital(PB)-treatment on the metabolism of benzene in vitro and in vivo, and on the benzene-induced hemotoxicity, were investigated. Ethanol consumption markedly enhanced in vitro metabolism of both benzene and phenol in rat liver, whereas PB-treatment, which enhanced the metabolism of phenol to some degree (about one-third of ethanol-induced enhancement), did not affect the metabolism of benzene. In a single exposure experiment with rats, ethanol increased benzene metabolism in vivo as evidenced by accelerated disappearance of benzene from the blood as well as by elevated urinary excretion of phenol, whereas PB produced little or no significant influence on the metabolism. In a 3-week exposure experiment, ethanol administration accelerated benzene disappearance from the blood in agreement with the single exposure experiment, but it tended to decrease urinary phenol excretion with repetition of exposure, probably due to concomitant stimulation of subsequent phenol metabolism by ethanol. Again, PB-treatment produced only a negligible effect on the metabolism of benzene. Ethanol consumption aggravated benzene-induced hemopoietic disorder as evidenced by a marked decrease in the peripheral white blood cell number. PB produced a protective effect on the toxicity. It is concluded that ethanol potentiates benzene toxicity by accelerating (1) hydroxylation of benzene, a rate-limiting step of benzene metabolism and (2) transformation of phenol into highly toxic metabolites.     

The effect of benzene on the activity of adenosine deaminase in tissues of rats

Swiss Albino (Rat rattus norvegicus) rats were intraperitoneally injected with a 100 mg kg(-1) dosage of benzene, a toxic and carcinogenic agent widely used for industrial purposes. Changes in the adenosine deaminase (ADA) activity in the liver, kidney and serum of rats were investigated at 0, 2, 4, 8, 16, 32 and 64 h following injection. Serum physiological was administered to each control group. Enzyme activities were measured spectrophotometrically. Our purpose was to further investigations of some diseases caused by benzene, and present evidence of variations in the activity of ADA enzyme effected by benzene. While benzene caused significant inhibitions in ADA activity in the liver at 16 and 32 h and at 0.05 probability level, no significant inhibition or activation occurred at other test periods (hours). ADA activity did not present any significant variation in the kidneys. It was observed that ADA activity displayed similar patterns in the control groups. Comparisons of ADA activities in the two groups showed a statistically significant decrease between 4(th) and 64(th) hours (p< 0.05), demonstrating a direct correlation between benzene and its effects on ADA enzymes.     

Sex differences in oxidative stress induced by benzene in rats

Role of sex differences on oxidative stress induced by benzene has been studied in liver, kidney and lungs of rat. It was observed that benzene administration enhanced lipid peroxidation in liver, kidney and lungs of rat, nevertheless, significant variations were recorded in male and female rats. Decrease of GSH and CYTP(450)2E1 was higher in female rats than male rats except lungs. The results suggest that oxidative stress induced by benzene is higher in female rats.     

The inhibition of mitochondrial DNA replication in vitro by the metabolites of benzene, hydroquinone and p-benzoquinone

Rat liver mitochondria incubated with the metabolites of benzene, p-benzoquinone or 1,2,4-benzenetriol, showed a dose-dependent inhibition of [3H]dTTP incorporation into mtDNA with median inhibitory concentrations of 1 mM for each compound. Benzene and the metabolites phenol, catechol and hydroquinone did not inhibit at concentrations up to 10 mM. Similarly, incubation of p-benzoquinone or hydroquinone with rabbit bone marrow mitochondria showed a dose-dependent inhibition of mtDNA synthesis with 50% inhibition at 1 mM and 10 mM, respectively. That these metabolites inhibit mitochondrial replication was evidenced by the fact that [3H]dTTP incorporation into characteristic 38S, 27S and 7S mitochondrial replication intermediates was decreased by the quinones, as analyzed on 5-20% neutral sucrose velocity gradients. p-Benzoquinone, hydroquinone and 1,2,4-benzenetriol inhibited the activity of partially purified rat liver mtDNA polymerase gamma using either activated calf thymus DNA or poly(rA) X p(dT)12-18 as primer/template, with 50% inhibitory concentrations of 25 microM, 25 microM and 180 microM, respectively. Preincubation of the metabolites with polymerase gamma or primer/template, followed by removal of the unreacted metabolite by gel filtration, indicated that inhibition resulted from interaction of the metabolites with the enzyme, rather than with the template. Binding appeared to involve a sulfhydryl residue on the enzyme since the binding of [14C]hydroquinone was prevented by N-ethylmaleimide. The ability of hydroquinone or p-benzoquinone to inhibit binding of [14C]hydroquinone to the enzyme suggests that the compounds bind to a common site or are converted to a common intermediate. Inhibition of, or changes in, replication in mitochondria of bone marrow cells by hydroquinone and p-benzoquinone may explain the changes in the mitochondrial genome observed in marrow stem cells in acute myelogenous leukemia and may suggest a mechanism for benzene leukemogenesis.     

Genotoxicity of two metabolites of benzene: phenol and hydroquinone show strong synergistic effects in vivo

Possible interactions between hydroquinone (HQ) and phenol (PHE), 2 known benzene metabolites, in inducing micronuclei in mouse bone marrow cells were investigated. HQ and PHE administered alone gave weak and negative results, respectively, at the doses tested. However, simultaneous administration of both compounds caused a considerable increase in the induction of micronuclei as well as an increase in bone marrow toxicity. Using 3 different statistical methods, it was shown that the observed joint effect was significantly higher than additive interaction, and was close to multiplicative interaction. These findings bring further support to the hypothesis that the toxic and genotoxic effects of benzene are produced by several metabolites acting synergistically.     

Benzene and the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depression in mouse bone marrow cells

Benzene (880 mg/kg) and 4 of its metabolites, i.e., phenol (265 mg/kg), hydroquinone (80 mg/kg), catechol (40 mg/kg), and p-benzoquinone (5-20 mg/kg) have been tested for their capability to induce micronuclei in bone marrow cells of male mice after oral administration or intraperitoneal injection. Oral administration of benzene shows more activity than intraperitoneal injection, whereas the metabolites show more activity if administered by the latter method. The respective genotoxic strengths of the benzene metabolites are the following: hydroquinone much greater than phenol greater than catechol = p-benzoquinone. This last is active when administered orally.     

A high pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

The glucuronide and sulfate conjugates of benzene metabolites as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl [14C]glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount of metabolite present in urine following exposure to [3H]benzene was determined using p-nitrophenyl [14C]glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm [3H]benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. Phenylsulfate, muconic acid, and pre-phenylmercapturic acids as well as an unknown with a HPLC retention time of 7 min were the major metabolites in the liver. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.     

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity,number of granulopoietic stem cells and frequency of micronuclei in mice

The effects of benzene and benzene metabolites (hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.     

Modulation of CYP4502E1 and oxidative stress by testosterone in liver and kidney of benzene treated rats

Bilateral castration increased lipid peroxidation and consequently reduced glutathione in both liver and kidney. Testosterone administration reduced lipid peroxidation in the liver of castrated and benzene treated rats, however, reduced glutathione status could not be restored. Benzene depleted CYP4502E1 in castrated rats, however, the enzyme was restored in liver and kidney both after testosterone treatment. The results suggest that testosterone affects the metabolism and disposition of benzene by influencing CYP4502E1. Other hormonal and cellular/molecular factors may also alter the actions of testosterone. Testosterone dependent mechanism of toxicity of benzene in the liver and kidney has been discussed.     

Toxicogenomic analysis of gene expression changes in rat liver after a 28-day oral benzene exposure

Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.     

Dose-dependent reduction of 3,2'-dimethyl-4-aminobiphenyl-derived DNA adducts in colon and liver of rats administered celecoxib

Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated (32)P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N(2)-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.     

The high mobility group of nuclear proteins as biomarkers of age and caloric restriction in rats.

The quantitative levels and phosphorylation states of the high mobility group (HMG) of proteins were investigated in bone marrow, brain, heart, kidney, liver, pancreas, spleen, testis and thymus of three groups of male Fischer 344 rats. Two groups of rats, young ad libitum (Y/AL - 1 1/2 mo.) and old ad libitum (O/AL - 28 mo.), had free access to rat chow, and a third group of old rats were maintained on a caloric restricted intake (O/CR - 28 mo.). The quantities of HMGs 1,2,14 and 17 were significantly reduced in O/AL rats compared with Y/AL rats in all tissues examined, and in many cases, the amount of HMGs of O/CR rats were increased by varying degrees from O/AL animals. In G2-phase nuclei of bone marrow, spleen and testis, phosphorylation of HMG proteins was reduced significantly in O/AL rats, but was enhanced in O/CR animals (especially HMG14). These levels of HMGs in O/CR animals, altered by age and diet dependent factors, reflect a condition which is more reminiscent of Y/AL than O/AL animals.     

Benzene transformation in nitrifying batch cultures

The effect of benzene on the nitrifying activity of a sludge produced in steady-state nitrification was evaluated in batch cultures. Benzene at 10 mg/L inhibited nitrate formation by 53%, whereas at 5 mg/L there was no inhibition. For initial benzene concentrations of 0, 7, and 10 mg/L, the specific rates of NO(3)(-)-N production were 0.545 +/- 0.101, 0.306 +/- 0.024, and 0.141 +/- 0.010 g NO(3)(-)-N/g microbial protein-N.h, respectively. The specific rates of benzene consumption at 7, 12, and 20 mg/L were 0.034 +/- 0.003, 0.050 +/- 0.006, and 0.027 +/- 0.002 g/g microbial protein-N.h, respectively. Up to a concentration of 10 mg/L, benzene was first oxidized to phenol, which was later totally oxidized to acetate. Benzene at higher concentrations (20 and 30 mg/L) was converted to intermediates other than acetate, phenol, or catechol. These results suggest that this type of nitrifying consortium coupled with a denitrification system may have promising applications for complete removal of nitrogen and benzene from wastewaters.     

Methyl-substitution of benzene and toluene in preparations of human bone marrow

The metabolism of benzene and toluene was investigated in preparations of human bone marrow incubated with S-adenosyl-L-methionine. Benzene undergoes a methyl-substitution reaction to yield toluene as a metabolite. Furthermore, toluene undergoes methyl-substitution in preparations of human bone marrow incubated with S-adenosyl-L-methionine to yield o-xylene, m-xylene, and p-xylene. Metabolites were detected by gas chromatography and mass spectroscopy. No metabolism of either benzene or toluene was detected when a boiled bone marrow preparation was used in the incubation, demonstrating the enzymatic nature of the S-adenosyl-L-methionine dependent methylation of both benzene and toluene.