Donald E. Nerland

Inhibition of erythropoiesis by benzene and benzene metabolites

Mice were injected sc with benzene or one of its metabolites, phenol, catechol, or hydroquinone. The ability of these compound to inhibit erythropoiesis was quantified by measuring the incorporation of 59Fe into developing erythrocytes. Benzene decreased 59Fe incorporation into developing erythrocytes in a dose-dependent manner. Maximum inhibition was observed when benzene was administered 48 hr prior to initiation of the 59Fe uptake test. The three metabolites of benzene also significantly inhibited 59Fe incorporation when they were administered 48 hr prior to initiation of 59Fe uptake assay. The degree of inhibition observed with the metabolites was not as great as that observed with benzene. Coadministration of the microsomal mixed-function oxidase inhibitor, 3-amino-1,2,4-triazole, abolished the erythropoietic toxicity of benzene and phenol but had no effect on the catechol- or hydroquinone-induced toxicity.

The Antioxidant/electrophile Response Element Motif

Cells exposed to oxidative stress or electrophilic xenobiotics respond by transcriptionally up-regulating a battery of genes that contain a cis-acting element in their promoter region known as the antioxidant/electrophile response element (ARE). Mutational analysis of the promoter regions of ARE-containing genes led to the creation of two different models for the ARE; a core ARE (cARE: RTGACnnnGC) and an extended ARE (eARE: TMAnnRTGAYnnnGCAwwww). Using bioinformatic software we have aligned the promoter regions of several ARE-containing genes to produce two position-specific probability matrices that independently describe the cARE and eARE. These matrices can also be used to quantitatively assess putative AREs

Tissue distribution of N-acetyltransferase 1 and 2 catalyzing the N-acetylation of 4-aminobiphenyl and O-acetylation of N-hydroxy-4-aminobiphenyl in the congenic rapid and slow acetylator Syrian hamster.

N‐acetyltransferase 1 (NAT1) and 2 (NAT2) enzymes catalyzing both deactivation (N‐acetylation) and activation (O‐acetylation) of arylamine carcinogens such as 4‐aminobiphenyl (ABP) were investigated in a Syrian hamster model congenic at the NAT2 locus. NAT2 catalytic activities (measured with p‐aminobenzoic acid) were significantly (P < 0.001) higher in rapid than slow acetylators in all tissues (except heart and prostate where activity was undetectable in slow acetylators). NAT1 catalytic activities (measured with sulfamethazine) were low but detectable in most tissues tested and did not differ significantly between rapid and slow acetylators. ABP N‐acetyltransferase activity was detected in all tissues of rapid acetylators but was below the limit of detection in all tissues of slow acetylators except liver where it was about 15‐fold lower than rapid acetylators. ABP N‐acetyltransferase activities correlated with NAT2 activities (r2 = 0.871; P < 0.0001) but not with NAT1 activities (r2 = 0.132; P > 0.05). Levels of N‐hydroxy‐ABP O‐acetyltransferase activities were significantly (P < 0.05) higher in rapid than slow acetylator cytosols for many but not all tissues. The N‐hydroxy‐ABP O‐acetyltransferase activities correlated with ABP N‐acetyltransferase activities (r2 = 0.695; P < 0.0001) and NAT2 activities (r2 = 0.521, P < 0.0001) but not with NAT1 activities (r2 = 0.115; P > 0.05). The results suggest widespread tissue distribution of both NAT1 and NAT2, which catalyzes both N‐ and O‐acetylation. These conclusions are important for interpretation of molecular epidemiological investigations into the role of N‐acetyltransferase polymorphisms in various diseases including cancer.

Acute acrylonitrile toxicity: studies on the mechanism of the antidotal effect of D- and L-cysteine and their N-acetyl derivatives in the rat.

Thiol-containing antidotes for acute acrylonitrile (AN) toxicity may exert their action by chemically reacting with AN, by replacing critical sulfhydryl groups cyanoethylated by AN, and by detoxifying cyanide produced from AN metabolism. We have evaluated the ability of the optical isomers of cysteine and N-acetylcysteine to act as antidotes against AN toxicity in order to assess the relative importance of each of these three antidotal mechanisms. The toxicity of AN was determined in male Sprague-Dawley rats and compared to the toxicity determined after treatment with 2 mmol/kg of thiol antidote by computing a protective index (median lethal dose with antidote/median lethal dose without antidote). The protective indices of L-cysteine, D-cysteine, N-acetyl-L-cysteine, and N-acetyl-D-cysteine were 2.03, 1.97, 1.76, and 1.25, respectively. Measurements of urinary mercapturates, derived from the non-oxidative pathway of AN metabolism, indicated that none of the antidotes was able to significantly increase the excretion of these metabolites. Blood cyanide generated from the oxidative metabolism of AN and butyronitrile was also determined. All of the antidotes, except N-acetyl-D-cysteine, lowered blood cyanide levels. A comparison of these results with the predicted relative abilities of the enantiomers to participate in each of the three antidotal mechanisms leads to the conclusion that, under these experimental conditions, the best correlation exists with the cyanide detoxification mechanism.

Hematotoxicity and concentration-dependent conjugation of phenol in mice following inhalation exposure to benzene

Benzene is metabolized to one or more hematotoxic species. Saturation of benzene metabolism could limit the production of toxic species. Saturation of phase II enzymes involved in the conjugation of the phenolic metabolites of benzene also could affect the hematotoxicity of benzene. To investigate the latter possibility, we exposed male Swiss mice, via the inhalation route, to various concentrations of benzene for 6 h per day for 5 days. Following termination of the final exposure the mice were killed and the levels of phenylsulfate and phenylglucuronide in the blood determined. Spleen weights were recorded and the number of white blood cells counted. At low benzene exposure concentrations phenylsulfate is the major conjugated form of phenol in the blood. At high exposure concentrations, phenylglucuronide is the predominant species. The reductions in spleen weight and white blood cell numbers correlated with the concentration of phenylsulfate in the blood, but are most probably not causally related.

Induction of mouse liver glutathione S-transferase by ethanol

The induction of hepatic glutathione S-transferase by ethanol was investigated in male Swiss-Webster mice using a liquid diet. After a 7-day feeding period, mice that received 18, 27 or 36% of their calories as ethanol exhibited significant increases in the specific activity of glutathione S-transferase when 1,2-dichloro-4-nitrobenzene (DCNB), p-nitrobenzylchloride (NBC) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENP) were used as substrates. The observed increases in activity appeared to be related to the concentration of ethanol in the diet. Thus, mice fed a diet with 36% of the calories as ethanol exhibited the greatest increase in specific activity (DCNB, 75%; NBC, 60%, ENP, 34%). Pair-fed mice demonstrated similar changes in enzymatic activity. A time-course study indicated a 4-day feeding period was not sufficient to elicit significant induction, but a significant increase was apparent by day 7. This increase was maintained or increased through day 14. By comparison, 0.5 mg of phenobarbital/ml of diet produced a greater increase in enzymatic activity (DCNB, 449%; NBC, 227%; ENP, 219%). These results suggest that ethanol does induce glutathione S-transferase, but it is a relatively poor inducer of this enzyme.

Regular ArticleDose Dependence of Covalent Binding of Acrylonitrile to Tissue Protein and Globin in Rats

The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02–0.95 mmol AN/kg). The slopes of the dose–response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood ⪢ kidney = liver > forestomach = brain > glandular stomach ⪢ muscle. Similar dose–response behavior was observed for globin total covalent binding and for globinN-(2-cyanoethyl)valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose–response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity.

INTERFERONS AND DRUG METABOLISM

ABSTRACT The cytochrome P-450 system is one of the major sites for metabolism of drugs in the liver. Several studies have shown that treatment of rats with inducers of alpha/beta interferon resulted in depression of the cytochrome P-450 system, and the metabolism of drugs by that system. In mice, the degree of depression of the cytochrome P-450 system correlated with the genetic ability of inbred strains of mice to produce alpha/beta interferon in response to an inducer. In humans, individuals vaccinated with agents that can induce alpha/beta interferon demonstrated enhanced retention of the drug theophylline, and at times even reached toxic levels. We have been able to show that induction of or passive transfer of actual exogenous gamma interferon resulted in depression of the cytochrome P-450 system. Prolonged retention of the drug phenytoin was also observed when gamma interferon was induced or passively transferred. Very recent studies using very large quantities of pure alpha interferon obtained from bacteria that have incorporated mammalian genes for interferon production have indicated that passive transfer of alpha/beta interferon can also inhibit the cytochrome P-450 system. These results suggest that the inhibition of the cytochrome P-450 system by interferon inducers or interferon may play some role in synergistic beneficial effects observed in clinical trials when both interferon and a chemotherapeutic agent are applied. We are currently carrying out studies to determine the mechanism and significance of these effects.

Inhibition of Interferon–Alpha/Beta Induction in L-929 Cells by Benzene and Benzene Metabolites

Murine L-929 cells were treated with benzene or a series of benzene metabolites, washed and then interferon-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid. Exposure of the cells to benzene or phenol, a monocyclic metabolite of benzene, did not affect interferon-alpha/beta induction. However, exposure of the cells to p-benzoquinone, hydroquinone or catechol, dihydroxy- and diketo-metabolites of benzene, resulted in a severe inhibition of interferon-alpha/beta production. There was no significant loss of viability of the cell cultures. Additional studies with p-benzoquinone indicated that inhibition of interferon-alpha/beta was reversible and could be abrogated by addition of reduced glutathione to the cell cultures.

[94] Measurement of effect of interferon on metabolism of diphenylhydantoin

Publisher Summary This chapter describes techniques for measuring the effects of interferon induction and passive transfer on the metabolism of a drug––diphenylhydantoin––that is detoxified by cytochrome P-450. The induction and passive transfer of interferons have been shown to inhibit cytochrome P-450 activity in the liver of rodents. Cytochrome P-450 is one of the major enzyme systems for the metabolism of lipophilic compounds. The inhibition of cytochrome P-450 by interferon has great implications in two major areas. One area is the activation of carcinogens by cytochrome P-450, as many polycyclic aromatic hydrocarbon carcinogens require activation to the final carcinogenic form by the cytochrome P-450 system. Inhibition of cytochrome P-450 activity by interferon could inhibit the activation of carcinogens. The second area of interest is the effects of interferon on concomitantly administered drugs that are metabolized and detoxified by cytochrome P-450. Inhibition of cytochrome P-450 activity by interferon could result in prolonged retention of drugs that could yield either detrimental or beneficial effects for the host. The inhibition of diphenylhydantoin metabolism by interferon (IFN) induction and passive transfer is one of the readily available techniques for demonstrating that inhibition of cytochrome P-450 activity by interferon actually affected drug levels.