Biomedical subjects

R F Novak

Publications and source records attributed to R F Novak.

At least 37 records · Page 2Linked to original sources

PubMed1996

Differential expression of glutathione S-transferase isoforms in compartments of the testis and segments of the epididymis of the rat.

Specific cell types of the mammalian testes demonstrate varying susceptibility to toxic insult by chemical agents. The mammalian testis is divided into two major compartments: seminiferous tubules, the site of spermatogenesis, and interstitium, which contains the Leydig cells. Glutathione S-transferase (GST) expression was examined in isolated compartments of the rat testis and in segments of the epididymis. Western blot analysis revealed the presence of GST class alpha, mu, and pi bands in each of the isolated compartments of the testis, and HPLC analysis of monomeric isoforms provided evidence for differential expression of multiple GST isoforms in testicular compartments. All major isoforms (e.g., forms 1, 2, 3, 4, 6, 7, 8, 9, and 11) were detected in the cytosol of whole testis. Isoform subunit 4 was the major form in the tubule, whereas isoform subunit 11 is the dominant form in the Leydig cells. Isoform subunits 3, 4, and 6 were enriched in the tubules as compared to interstitial or Leydig cells. The preferential action of reproductive toxicants at specific stages of aging may be due to an age-dependent expression of the activating or detoxifying enzymes in the reproductive tract. Therefore, the age-dependent expression of testicular GST isoforms was also examined. Expression of isoform subunits 2 and 4 displayed an age dependence, with the largest increase in these subunits occurring between ages 4 and 15 weeks. Isoform expression did not correlate with serum testosterone levels. HPLC analysis of the GST isoforms in the longitudinal segments of the epididymis and vas deferens revealed differential expression within these segments. Total GST protein and catalytic activity was highest in the caput epididymis and progressively decreased toward the vas deferens. Isoform subunit 2 was the major form expressed in the epididymis. The results of this study indicate that the GSTs are differentially expressed in testicular compartments and epididymal segments, and that this may contribute to susceptibility of different cell types to xenobiotic damage.

Animals↗

PubMed1996

Effects of hydrazine, phenelzine, and hydralazine treatment on rat hepatic and renal drug-metabolizing enzyme expression.

The hepatic and renal toxicity associated with hydrazine treatment has been linked to free radical damage resulting from oxidative metabolism by cytochrome P4502E1 (CYP2E1). Despite this association, there has been little characterization of the effects of hydrazine treatment on the expression of hepatic and renal CYP2E1 or glutathione-S-transferase-alpha (GST-alpha), an enzyme responsible for catalyzing the conjugation of free radicals with reduced glutathione. Therefore, the effects of treatment with hydrazine or one of the therapeutic hydrazines phenelzine and hydralazine on rat hepatic and renal CYP2E1 and GST-alpha expression were investigated. Adult male Sprague-Dawley rats were treated with 0.9% saline vehicle (1 dose ip), hydrazine (100 mg/kg ip), phenelzine (100 mg/kg ip), or hydralazine (25 mg/kg ip). CYP2E1 mRNA and protein levels were monitored by Northern and immunoblot analyses, respectively, and GST-alpha Ya and Yc subunit levels were determined by immunoblot analysis. Hydralazine administration caused a significant (approximately 159%) increase in renal GST-alpha subunit expression. In addition, hydrazine and phenelzine treatment produced substantial elevations (approximately 464% and 566%, respectively) in renal CYP2E1 protein, whereas hydralazine administration did not alter renal CYP2E1 expression. Changes in rat hepatic GST-alpha Ya or Yc subunit levels after treatment with hydrazines phenelzine, or hydralazine were not statistically significant. Similarly, hepatic CYP2E1 levels were not significantly altered after treatment with hydrazine, phenelzine, or hydralazine. Northern blot analysis revealed that the observed increases in renal CYP2E1 protein levels after treatment with hydrazine or phenelzine were not accompanied by concomitant increases in CYP2E1 mRNA. These results suggest that treatment with hydrazine or the therapeutic hydrazine phenelzine significantly increases the expression of rat renal CYP2E1 protein, and that the molecular mechanism responsible for these effects are posttranscriptional in nature.

Animals↗

PubMed1996

Cytochrome P4502E1- and cytochrome P4502B1/2B2-catalyzed carbon tetrachloride metabolism: effects on signal transduction as demonstrated by altered immediate-early (c-Fos and c-Jun) gene expression and nuclear AP-1 and NF-kappa B transcription factor levels.

We have previously reported that carbon tetrachloride (CCI4) stimulates c-fos, c-jun, and Ca(2+)-activated neutral protease gene expression in rat hepatic tissue (Zawaski et al., Biochem. Biophys. Res. Comm. 197, 585-590, 1993). The proteins c-Fos and c-Jun constitute inducible transcription factors in signal transduction and regulate the transcriptional activation of a battery of genes involved in cell growth and division. The present study was initiated to characterize the role of cytochrome P450 expression and metabolic activation on the magnitude of immediate-early (i.e. c-fos and c-jun) gene expression. Animals were treated either with diallyl sulfide, N-acetylcysteine, pyridine, or phenobarbital before treatment with CCI4. Total and poly(A)+ RNA were isolated, and c-fos and c-jun mRNA levels were analyzed by Northern blot and reverse transcriptase-polymerase chain reaction analyses. Treatment of animals with CCI4 increased c-fos and c-jun mRNA levels from below the limit of detection in control tissue to intense bands within 30 min of treatment, with maximal expression monitored at 1 and 2 hr posttreatment. Treatment of animals with diallyl sulfide alone also elevated c-fos and c-jun mRNA expression to detectable levels. However, pretreatment of animals with diallyl sulfide before treatment with CCI4 produced a 76-92% decrease in c-fos and c-jun mRNA levels, relative to that monitored for CCI4-treated animals. Pretreatment with N-acetylcysteine did not affect c-fos or c-jun mRNA levels and diminished CCI4-stimulated c-fos and c-jun gene expression by 44 and 55%, respectively, relative to the immediate-early gene mRNA levels monitored in the hepatic tissue of CCI4-treated animals. Pretreatment of animals with the CYP2E1 inducer pyridine for 24 hr had only a marginal effect on c-fos mRNA levels, but increased CCI4-stimulated c-fos and c-jun mRNA levels by an additional approximately 2- to approximately 4-fold over those monitored in the uninduced hepatic tissue of CCI4-treated animals. Whereas phenobarbital treatment alone enhanced c-fos expression only marginally, CCI4 treatment of phenobarbital-pretreated animals increased c-fos expression by up to an additional approximately 8-fold and c-jun mRNA levels by up to an additional approximately 5-fold over the respective levels monitored in the hepatic tissue of CCI4-treated animals. Enhanced CYP2E1 or CYP2B1/2B2 levels after treatment with pyridine or phenobarbital elevated c-fos mRNA over untreated controls. This increase was marginal, however, and detectable only with reverse transcriptase-polymerase chain reaction. Examination of nuclear levels of the heterodimeric c-Fos and c-Jun AP-1 transcription factor complex revealed a time-dependent increase in AP-1 levels. AP-1 transcription factor binding was confirmed using competitor consensus sequences and antibody supershifts. Nuclear levels of NF-kappa B, a transcription factor complex implicated in hepatocyte proliferation and apoptotic or programmed cell death, were also examined. NF-kappa B, which consists of the p50 and p65/Rel A polypeptides, was increased in hepatic nuclear extracts at 2 and 24 hr after CCI4 administration, with a concomitant decrease in the p50 polypeptide. Thus, the magnitude of CCI4 stimulation of the immediate-early genes c-fos and c-jun is dependent on metabolic activation by the P450s, and the magnitude of the effect is dependent on the levels and isozyme composition of P450s in the tissue. Furthermore, nuclear transcription factor levels of AP-1 and NF-kappa B are elevated in response to this toxicant.

Animals↗

PubMed1995

Pyridine induction of Sprague-Dawley rat renal cytochrome P4502E1: immunohistochemical localization and quantitation.

Previous research has shown that i.p. injection of rats with pyridine results in a significant increase in immunoreactive renal cytochrome P4502E1 (alcohol-inducible form) in a dose- and time-dependent manner. However, the cellular location of renal P4502E1 in rats was not reported. Thus, it was not known whether the pyridine-induced increase in renal P4502E1 resulted from increased production of the enzyme in cells which normally express P4502E1 or from de novo expression in cells normally devoid of the protein. To address these questions, rats were injected i.p. with either 200 mg pyridine/kg body wt./day for 1, 2, 3, or 4 days (n = 2/group) or injected once with an equal volume of sterile, pyrogen-free saline (control group; n = 2). Kidney tissue samples from saline- and pyridine-exposed rats were processed by light microscopy and were immunochemically stained to detect rat cytochrome P4502E1. Most of the immunoreactive P4502E1 was located within renal cortical epithelial cells lining proximal and distal tubules of the cortex with lesser--but consistent--amounts present in tubular epithelial cells within the inner and outer medulla. Pyridine exposure resulted in a 2-3-fold increase in P4502E1 immunoreactivity in proximal cortical tubules surrounding glomeruli and cortical blood vessels. The results of this study demonstrate a cell-specific distribution of cytochrome P4502E1 within the rat kidney and indicate that pyridine exposure results in a selective induction of immunoreactive P4502E1 in tubule epithelial cells which constitutively express this enzyme. The results of this study provide a morphologic basis for interpreting cell-specific nephrotoxicity due to xenobiotics that are biotransformed to toxic metabolites by renal P4502E1.

Animals↗

PubMed1995

Gender-specific and developmental differences in protein kinase C isozyme expression in rat liver.

Protein kinase C (PKC) activity is important in regulating cellular growth and differentiation and is believed to play a role in the promotional stage of carcinogenesis. To determine the effects of age and gender on PKC expression, isozyme-specific antibodies and immunoblot analyses were employed to examine the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon in the cytosolic and membrane fractions isolated from hepatic tissue of 1, 3, 5 and 12 week old male or female rats. PKC alpha levels were comparable at 1 week of age in the respective male and female hepatic fractions. In contrast, at 3, 5 and 12 weeks of age, cytosolic PKC alpha levels were approximately 2-, 5- and 7-fold greater, respectively, in females than in males. At 5 weeks of age, cytosolic levels of PKC delta were approximately 2-fold higher in females than in males. Other PKC isozymes were either below the limit of detection (PKC gamma), or failed to exhibit any gender-related differences (PKC beta and PKC epsilon). At 12 weeks of age, PKC activity was 1.7- or 2.4-fold greater in hepatic cytosol and membrane fractions, respectively, from females than in male samples. These results show distinct gender-specific and developmental differences in hepatic PKC isozyme expression, which may play a role in susceptibility to cancer.

Aging↗

PubMed1995

3-Methylcholanthrene and pyridine effects on CYP1A1 and CYP1A2 expression in rat renal tissue.

The effects of 3-methylcholanthrene (3-MC) and pyridine on rat renal cytochrome P450 (CYP) 1A1 and 1A2 mRNA expression have been examined by Northern-blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Southern-blot analysis of the PCR products. Northern-blot hybridization and RT-PCR analyses of kidney poly(A)+ RNA revealed that 3-MC treatment produced a time-dependent increase in the renal CYP1A1 and 1A2 mRNA levels, with CYP1A1 and 1A2 mRNA levels maximally increased at 24 and 18 hr, respectively, after treatment. These data were confirmed via RT-PCR analysis using a subsaturating level of cDNA template and by Southern-blot analysis of the PCR products. This approach served as the foundation for examining the effects of pyridine on CYP1A1 and 1A2 expression in renal tissue. RT-PCR analysis of renal CYP1A1 and 1A2 poly(A)+ RNA levels after treatment with pyridine (200 mg/kg/day for 3 consecutive days) revealed that CYP1A1 mRNA levels were maximally elevated approximately 10-fold after pyridine treatment for 2 consecutive days, whereas CYP1A2 mRNA levels were maximally elevated approximately 3-fold at 24 hr after treatment. The mRNA levels of glyceraldehyde 3-phosphate dehydrogenase, which served as an internal control, remained constant after 3-MC or pyridine treatment. These results show that expression of CYP1A1 and 1A2 mRNAs is enhanced in renal tissue after exposure to 3-MC or pyridine, and that constitutive expression of CYP1A1 seems to be greater than that of CYP1A2 in renal tissue.

Animals↗

PubMed1995

Glutathione S-transferases and gamma-glutamyl transpeptidase in the rat nervous systems: a basis for differential susceptibility to neurotoxicants.

Glutathione and its related enzymes play a major role in the detoxification of toxic chemicals. In rat brain the pattern of distribution of reduced glutathione exhibits cellular heterogeneity, suggesting also the possibility of cellular differences in glutathione conjugating capacity. To understand the potential role of GSH in detoxification of neurotoxicants, the distributions of the glutathione conjugating and metabolizing enzymes, glutathione S-transferase (GST; alpha-, mu- and pi-classes) and gamma-glutamyl transpeptidase (gamma-GT) were determined immunohistochemically in brain, lumbar spinal cord and dorsal root ganglia (DRG) of adult Sprague-Dawley rats using polyclonal antibodies. The influence of tissue fixation on apparent distribution was also examined. Glial cells and neurons throughout the nervous system were only weakly positive with alpha-GST in frozen sections. No immunoreactivity for the alpha-class GSTs was observed in any of the paraformaldehyde-fixed neural specimens examined. In microwave-fixed frozen sections, immunoreactivity to mu-GST was found in astrocytes and neurons throughout the brain and spinal cord, and in the neurons and satellite cells of the DRG. Immunoreactivity for pi-GST was seen in oligodendrocytes but not in astrocytes in any region of the CNS examined. Similarly, satellite cells of the DRG were positive for pi-GST. Neuronal perikarya of the entire neopallium, hippocampus, cerebellum, brainstem, spinal cord and DRG were also positively stained for pi-GST. The differential staining of astrocytes and oligodendrocytes with pi- and mu-GST was unaltered in paraformaldehyde fixed tissues, but the neuronal immunostaining was lost. The ependyma, pia and choroid plexus stained positively with all three GST antibodies regardless of fixation. Gamma-Glutamyl transpeptidase-like immunoreactivity was confined to non-neuronal elements of both central and peripheral nervous systems. Ependymal cells throughout the central nervous systems stained intensely with antibodies directed against gamma-GT. Satellite and Schwann cells of the DRG and glial cells of the spinal cord and brain exhibited moderate to intense immunoreactivity for gamma-GT. The heterogeneous cellular distribution of glutathione and its metabolizing enzymes may reflect cellular differences in capacity for metabolic processing of both endogenous compound and xenobiotics.

3,3'-Diaminobenzidine↗

PubMed1995

Induction of nasal carboxylesterase in F344 rats following inhalation exposure to pyridine.

Carboxylesterases (CEs) in the nasal mucosa metabolize some inhaled esters, including industrially important acrylates and acetates, to toxic acid metabolites that produce site-specific lesions in the nasal epithelium. The metabolic capacity of CEs in the normal nasal mucosa is theoretically sufficient to protect the lower respiratory tract from toxicant-induced injury at concentrations of acrylates and acetates likely to be inhaled in industrial environments. Thus, alterations in the metabolism and toxicity of these substrates would be predicted with changes in the amount or activity of CE in the nasal mucosa. Although many other nasal enzymes have been reported to be relatively refractory to induction, the amount of CE in the nasal mucosa can be increased by inhalant exposure. In the liver, expression of CEs may be elevated in response to exposure to P450 inducers. To examine this phenomenon in the nose with the widely used industrial solvent pyridine, we examined the effect of pyridine inhalation at the threshold limit value concentration of 5 ppm, or at 444 ppm, 6 hr/day for 4 days on the localization and amount of immunoreactive CE in olfactory mucosas of F344/N rats. CE immunoreactivity was increased in Bowman's glands following exposure to 5 or 444 ppm pyridine, and in sustentacular cells most notably following the 5 ppm exposure. Quantitative densitometry showed a statistically significant, dose-related increase in the density of immunoreactive CE in Bowman's glands of pyridine-exposed rats. These results indicate pyridine, and possibly other solvents, can induce nasal CE, an enzyme not directly involved in the metabolism of those solvents, following low-dose, short-term exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗

PubMed1995

Xenobiotic-enhanced expression of cytochrome P450 2E1 and 2B1/2B2 in primary cultured rat hepatocytes.

Investigation of the posttranscriptional mechanisms involved in the xenobiotic-mediated enhancement of cytochrome P450 2E1 (CYP2E1) expression has been limited by a lack of a functional primary hepatocyte cell culture system. We examined the effects of ciprofibrate (CIPRO) and pyridine (PYR) treatment on the expression of CYP2E1, P450 4A (CYP4A), and P450 2B (CYP2B) in primary rat hepatocytes cultured on Vitrogen or Matrigel substratum and in the presence of Chee's medium. Cells were cultured for 72 hr or longer before initiation of treatment. Northern blot analyses indicated that 24-hr CIPRO treatment enhanced the expression of CYP4A, and CYP2B mRNA in a concentration-dependent manner, with maximal induction of CYP2E1 mRNA (2- to 3-fold) and CYP4A1 mRNA (up to approximately 15-fold) monitored at 30-300 microM CIPRO. Maximal CYP2B mRNA levels (7- to 8-fold) were monitored at 300-1000 microM CIPRO. Treatment of hepatocytes for 24, 48, and 72 hr with 30 microM CIPRO showed progressive increases in CYP2B and CYP4A mRNA levels, with approximately 13- and 60-fold elevations in the respective mRNAs occurring at 72 hr posttreatment. In contrast, CYP2E1 mRNA levels were maximally elevated between 2- and 3-fold at both 24 and 48 hr and were returning to basal levels by 72 hr. Western blot analyses revealed that 24-hr PYR (25 mM) treatment of CIPRO-treated cells, in the absence of any further increase in CYP2E1 mRNA levels, increased CYP2E1 protein levels approximately 6- to 8-fold. PYR treatment also increased CYP2B mRNA and CYP2B1/2B2 protein levels approximately 16-fold relative to cells treated only with CIPRO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA.

Poly(A) tail length is important in the stability and translation of mRNA. We describe procedures for the rapid and reproducible analysis of poly(A) tail length of a single mRNA species contained in a sample of total hepatic RNA. A short 3' fragment of a specific mRNA is prepared by RNase H digestion of the targeted mRNA region annealed to a short DNA oligonucleotide. The length of the poly(A) tail of the 3' fragment is then determined by running the sample on a polyacrylamide gel, by electrophoretic transfer, by probing with a radiolabeled cDNA and by comparing the size of the detected region with a specific RNA ladder or a DNA ladder.

Animals↗

PubMed1995

Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system.

The distribution of glutathione (GSH) and glutathione S-transferases (GSTs) in the adult rat brain is cell-type specific, but their cellular distribution in the developing central nervous system is unknown. In the present study, GSH distribution in the mouse nervous system was visualized by mercury orange histochemistry and class-specific GSTs were localized by immunohistochemistry at ages E13 to PN30. Both neuronal and glial progenitor cells stain uniformly positive for GSH at E13. Spinal anterior horn neurons become GSH-negative by E17, at which time neurons and glia in other CNS regions are still GSH-positive. By PN5, most neurons have lost GSH staining and are surrounded by GSH-rich neuropil, ependyma, and vasculature. Olfactory mitral and granule cells, cerebellar granule cells, and dorsal root ganglion (DRG) neurons retain consistently high levels of GSH throughout development and into adulthood. Immunoreactivity to alpha-class GST antisera is not observed in the CNS until PN10, when very weak staining becomes apparent in the pia, ependyma, choroid plexus and neurons throughout the brain and spinal cord. Immunoreactivity to mu-GST is observed in neurons and astrocytes (but not oligodendrocytes), pia, ependyma, and choroid plexus throughout the brain by PN10. pi-GST immunoreactivity is observed in all cells of the embryonic nervous system. Postnatally, it is found in neurons and oligodendrocytes (but not astrocytes) in all regions of the brain and spinal cord as well as in pia, ependyma, and choroid plexus. The neurons and satellite cells of the DRG are immunoreactive to alpha-, mu-, and pi-GST antisera at all time points examined. The developmental changes in the cellular distribution of GSH and GSTs suggest that enzymatic conjugation and antioxidant activities may also be cell specific during brain development.

Animals↗

PubMed1994

Malotilate, a hepatoprotectant, suppresses CYP2E1 expression in rats.

The expression of CYP2E1 was examined in hepatic tissue from rats treated with malotilate (MT), a hepatoprotectant. Microsomal p-nitrophenol hydroxylase activity in MT-treated rats was decreased to 66% and 47% of control activity at day 2 and 3 post-treatment. SDS-PAGE and immunoblot analyses of hepatic microsomes prepared from MT-treated rats showed that CYP2E1 levels were decreased below the limit of detectability. In contrast, CYP2B1 levels were increased in MT-treated microsomes, as assessed by immunoblot analyses. MT, however, failed to modulate CYP1A expression. RNA hybridization analysis revealed that CYP2E1 mRNA levels failed to change significantly by day 2 or 3 post-treatment, whereas microsomal epoxide hydrolase mRNA levels were elevated approximately 3-fold at the same time points. These results demonstrate that MT effectively suppresses CYP2E1 expression in the absence of transcriptional inactivation.

Animals↗

PubMed1994

Inhibition of cytochrome P4502E1 expression by organosulfur compounds allylsulfide, allylmercaptan and allylmethylsulfide in rats.

Cytochrome P4502E1 (CYP2E1) is active in both detoxication and activation of small organic molecules. The effects of organosulfur compounds including allylsulfide (AS), allylmercaptan (AM) and allylmethylsulfide (AMS) on the expression of CYP2E1 were examined in rats. 4-Nitrophenol, aniline hydroxylase and N-nitrosodimethylamine demethylase activities, the rates of which represent the level of CYP2E1, decreased in hepatic microsomes isolated from rats treated with AS in a time-dependent manner by 45% to 90%, as compared to control. Pyrazine-induced hepatic microsomes exhibited approximately 5-fold increases in CYP2E1-catalysed metabolic activities, whereas the hepatic microsomes obtained after treatment of animals with both AS and pyrazine showed rates comparable to or less than those in control microsomes. AM or AMS suppressed constitutive and pyrazine-inducible levels of CYP2E1 similarly to AS. Immunoblot analyses of hepatic microsomes, using an anti-CYP2E1 antibody, showed that AS, AM and AMS significantly suppressed constitutive levels of CYP2E1 apoprotein after 24, 48 and 72 hr. Time-dependent induction of CYP2E1 by pyrazine was also completely blocked by treatment of animals with AS throughout the experimental period, as evidenced by immunoblot analysis. The levels of CYP2E1 apoprotein in the hepatic microsomes isolated from animals treated with both AM and pyrazine, or with both AMS and pyrazine were comparable to those in control hepatic microsomes at days 1-3 post-treatment. Treatment of rats with each of these organosulfur compounds caused no significant changes in the levels of CYP2E1 mRNA, as assessed by slot and northern blot analyses, suggesting that post-transcriptional regulation may be associated with the suppression of CYP2E1 apoprotein levels. The results of metabolic activities, immunoblot analyses and RNA blot analyses demonstrated that these organosulfur compounds are effective in suppressing constitutive and inducible expression of CYP2E1.

Allyl Compounds↗

PubMed1994

Hydrazine-mediated DNA damage: role of hemoprotein, electron transport, and organic free radicals.

The hydrazines represent an important class of xenobiotic agents encountered in the environment, in industrial settings, and in medical therapeutics. Agents with a hydrazine functionality are metabolized to toxic intermediates capable of damaging cellular macromolecules and stimulating proteolysis. Phenylhydrazine (PH), methylhydrazine (MH), hydrazine (HY), and the therapeutic agents phenelzine (PZ) and hydralazine (HD) were examined for their ability to undergo metabolism via HbO2 and to cause damage to added supercoiled phi x174 RF DNA. The hydrazines, when incubated in hemolysate, caused a time- and concentration-dependent strand scission of DNA as monitored using phi x174 RF DNA. The rank order for hydrazine-mediated damage was phenylhydrazine > phenelzine > hydrazine > hydralazine > methylhydrazine. In addition, hydrazine-mediated damage to DNA increased in proportion to protein concentration (i.e., HbO2 content) of the hemolysate. To examine whether the DNA damage resulted primarily from organic free radicals or reactive oxygen free radical species, a series of mechanistic studies employing antioxidants and a free radical scavenger was initiated. The antioxidants dimethylfuran, dimethyl sulfoxide, and dimethylthiourea failed to inhibit hydrazine-mediated DNA damage in hemolysate. In contrast, the free radical spin trap agent dimethylpyrrolidin-N-oxide effectively inhibited PH-mediated DNA damage, while the free radical scavenger N-acetylcysteine also showed a protective effect against PH-, PZ-, HD-, HY-, and MH-mediated DNA strand scission. Potassium ferricyanide-mediated methemoglobin formation and imidazole, a ligand for the heme moiety of hemoglobin, both inhibited PH-stimulated DNA damage in hemolysate demonstrating the importance of oxyhemoglobin to the process. These results suggest that organic free radicals play a dominant role, relative to oxygen free radical species, in hydrazine-mediated DNA strand scission.

DNA Damage↗

PubMed1994

Piperine effects on the expression of P4502E1, P4502B and P4501A in rat.

1. Treatment of rat with piperine (PIP) (1.4 mmol/kg, 3 days ip injections) resulted in an approximate two-fold increase in total liver microsomal P450 content relative to that in uninduced animals. 2. 4-Nitrophenol and aniline hyroxylase activities in the hepatic microsomes prepared from rat treated with PIP decreased by 30 and 28% respectively as compared with control. Immunoblot analyses also revealed decreased P4502E1 levels in hepatic microsomes from PIP-treated animals. 3. In contrast with P4502E1 suppression, hepatic 2B1 and 2B2 levels were significantly increased in PIP-induced animals, as evidence by both metabolic activity and immunoblot analysis of the liver microsomal fractions. The rate of hexobarbital hydroxylase activity in microsomes from PIP-treated animals was markedly elevated and was inhibited by approximately 62% in the presence of monoclonal anti-P4502B IgG. Immunoblot analyses demonstrated that P4502B1 and 2B2 levels in hepatic microsomes from PIP-treated animals were comparable with those from phenobarbital-treated animals. 4. 7-Ethoxycoumarin deethylase activity was elevated approximately two-fold in PIP-induced animals and was 17% of that derived from 3-methylcholanthrene-induced animals. 7-ethoxycoumarin deethylase activity in PIP-induced hepatic microsomes was inhibited 63% in the presence of monoclonal anti-P4501A antibody. Immunoblot analysis confirmed the increase in P4501A levels by PIP, which was 15% of that in hepatic microsomes from 3-methylcholanthrene-induced animals. 5. PIP treatment failed to affect microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GST) expression, as indicated by immunoblot analyses using polyclonal antibodies toward mEH and GST subunits Ya, Yb1, Yb2 and Yc. 6. These results demonstrate that PIP treatment suppressed P4502E1 expression and enhanced 2B and 1A expression, whereas this agent failed to affect hepatic mEH and GST expression.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1994

Regulation of cytochrome P-4501A and cytochrome P-4502E induction in the rat during the production of interferon alpha/beta.

The down regulation of constitutive hepatic microsomal cytochromes P-450 (P450) by interferons has been well described in experimental animals and humans, however the down regulation of induced forms of P450 has not been documented clearly. Differential down regulation of constitutive and induced P450 could alter the proportions of P450 enzymes and, hence, the relative bioactivation/detoxification of xenobiotics. We investigated the effects of polyinosinic acid-polycytidylic acid, a potent stimulator of interferon alpha/beta production on CYP1A and CYP2E induction in the rat. Polyinosinic acid-polycytidylic acid down regulated the constitutive and pyridine-induced expression of CYP2E1 and the pyridine- and beta-naphthoflavone-induced expression of CYP1A1 as demonstrated by metabolic activity and immunoblot analyses. Depression of CYP2E1 and CYP1A1 protein expression by polyinosinic acid-polycytidylic acid was accompanied by a corresponding decrease in mRNA encoding these proteins. Induction of CYP1A2 mRNA also was depressed. Therefore, interferon alpha/beta down regulated induction of members of the CYP1A and CYP2E subfamilies at a pretranslational level independent of the mechanism of induction. Induction of the CYP1A and CYP2E subfamilies did not confer resistance to down regulation by interferon, although the magnitude of down regulation by interferon appeared to be influenced by the magnitude of P450 induction. The potential significance of down regulation of induced P450 in the clearance of certain therapeutic agents and in xenobiotic bioactivation and detoxification is discussed.

Animals↗

PubMed1993

Evidence for enhanced expression of c-fos, c-jun, and the Ca(2+)-activated neutral protease in rat liver following carbon tetrachloride administration.

The effects of CCl4 on hepatic c-fos and c-jun gene expression were examined, and the correspondence between intermediate-early gene expression and the expression of the Ca(2+)-activated neutral proteinase (mu and mCANP) characterized. Administration of CCl4 to rats resulted in a pronounced dose- and time-dependent increase in c-jun and c-fos mRNA levels (approximately 8 to 17-fold) as detected by either Northern blot or RT-PCR analyses. The expression of mu and mCANP following CCl4 treatment was monitored by Northern blot analysis and mu and mCANP mRNA levels were found to be elevated approximately 5- to 15-fold over the time period of 18 to 24 h. Experiments were performed to determine whether the Fos-Jun heterodimeric AP-1 transcription factor complex bound the AP-1-like binding motif present in the mCANP gene. Gel retardation assays using hepatic nuclear extracts from CCl4-treated animals revealed binding of a protein to the AP-1-like motif present in the mCANP gene and competition and supershift assays confirmed the specificity of AP-1 transcription factor binding. These results show that CANP gene expression is enhanced in response to oxidative cellular damage and that the AP-1 complex may be involved in the regulation of CANP gene expression.

Animals↗

PubMed1993

Xenobiotic-modulated expression of hepatic glutathione S-transferase genes in primary rat hepatocyte culture.

CYP 2B1/B2 and 1A1 expression in primary rat hepatocytes plated on a substratum of Vitrogen using Chee's Essential Medium has been reported to be responsive to xenobiotic treatment (Jauregui, H.O., Ng, S.F., Gann, K.L. and Waxman, D.J. (1991) Xenobiotica 21, 1091-1106). Class alpha, mu and pi glutathione S-transferase (GST) gene expression in response to xenobiotic treatment using this primary hepatocyte culture system was examined and the results compared with those obtained for P4502B1/B2 and 1A1 expression. Cytosolic GST activity decreased approx. 75% during the first 48 h of culture relative to freshly isolated hepatocytes and subsequently, increased, attaining a level at 96 h that was 134% of the activity at 48 h post-plating. Treatment of the hepatocyte cultures with phenobarbital (2 mM) or 3-methylcholanthene (5 microM) for 24, 48, or 72 h, beginning 24 h after plating, resulted in significant increases in glutathione S-transferase activity relative to control, with maximal increases of 158 and 164% measured at 72 h following phenobarbital or 3-methylcholanthrene treatment, respectively. SDS-PAGE analysis of cytosolic proteins showed a substantial increase in the intensities of protein bands migrating in the region of the GSTs following phenobarbital, beta-naphthoflavone or 3-methylcholanthrene treatment. Immunoblot analysis of cytosolic fractions using affinity-purified class-specific GST IgGs confirmed that alpha, mu and pi-class GST isozymes were elevated approx. 1.5- to 2-fold following phenobarbital, or beta-naphthoflavone treatment; 3-methylcholanthrene was less effective in enhancing GST expression in cultured hepatocytes as compared to phenobarbital or beta-naphthoflavone. Although GST pi was below the limit of detection in freshly-isolated hepatocytes, enhanced expression of this form was observed in untreated hepatocytes cultured for longer than 72 h. Immunoblot analysis of microsomal fractions revealed that cytochrome P-4502B1/2B2 and 1A1 levels were increased significantly in hepatocyte cultures treated with phenobarbital or 3-methylcholanthrene, respectively, relative to the undetectable levels found in untreated controls. Northern blot analysis of poly(A)+ mRNA isolated from cultures that had been treated with phenobarbital or 3-methylcholanthrene showed an approx. 2- and 4-fold increase in the expression of alpha and pi class glutathione S-transferase mRNAs, respectively, as compared to untreated cells. The level of P-4501A1 or 2B1 mRNA was also markedly elevated following 3-methylcholanthrene or phenobarbital treatment, respectively. The results of this study demonor the first time, that expression of alpha, mu and pi-class glutathione S-transferase genes is effectively modulated in primary yet culture system by different classes of xenobiotics.

Animals↗