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K Randerath

Publications and source records attributed to K Randerath.

At least 37 records · Page 2Linked to original sources
PubMed1996

In vitro and in vivo (32)P-postlabeling analysis of 4-vinyl-1-cyclohexene (butadiene dimer) diepoxide-DNA adducts.

4-Vinyl-1-cyclohexene diepoxide (VCD), and industrial chemical, and its parent compound, 4-vinyl-1-cyclohexene (VCH), are potential health hazards, a they destroy oocytes in follicles in rodents. VCD is also a skin carcinogen at the site of application in both female and male mice and rats and after gavage, induces ovarian tumors in mice and forestomach tumors in rats. A (32)P-postlabeling assay was developed for the detection and measurement of VCD-DNA adducts. VCD, a direct-acting carcinogen, was reacted with DNA in vitro, as well as through mouse skin painting for 3 days with different doses of VCD. (32)P-Labeled adducts were separated by polyethyleneimine (PEI)-cellulose TLC and detected by screen-enhanced autoradiography. Comparable adduct profiles were obtained in vitro and in vivo. At higher doses (36-225 micro mol/mouse), adduct levels in vivo showed a linear dose response, while there was no difference between 14 and 36 micro mol/mouse. The limit of detection was estimated to be 1-3 adducts in 10(8) DNA nucleotides. The results show that VCD exposure gives rise to (presumably pre-mutagenic) DNA adducts in vivo and that (32)P-postlabeling can be applied to biomonitoring of VCD exposure.

Animals↗
PubMed1996

Tamoxifen metabolic activation: comparison of DNA adducts formed by microsomal and chemical activation of tamoxifen and 4-hydroxytamoxifen with DNA adducts formed in vivo.

One of our laboratories recently showed by 32P-postlabeling that administration of tamoxifen to mice induces two groups of hepatic DNA adducts comprising two major spots, nos. 3 and 5, respectively. 4-Hydroxytamoxifen and alpha-hydroxytamoxifen appear to be the proximate metabolites of groups I and II adducts, respectively. The relative significance of these two adduct groups for tamoxifen carcinogenicity remains to be established. To determine the activation mechanism(s) of tamoxifen and 4-hydroxytamoxifen, in vivo adducts were compared by 32P-postlabeling with adducts generated by microsomal or chemical activation in vitro. Microsomal activation of 4-hydroxytamoxifen and tamoxifen, respectively, in the presence of DNA and cumene hydroperoxide, induced two adducts, which mapped similarly to the corresponding in vivo adduct spots 3 and 5. Chemical oxidation of 4-hydroxytamoxifen with silver(II) oxide, followed by incubation of the product(s) with DNA, elicited the formation of a major spot (Q1), while tamoxifen itself did not react. Rechromatographic analyses revealed that in vitro fractions 3 and Q1 (from 4-hydroxytamoxifen) matched the major in vivo group I adduct fraction 3, consistent with the hypothesis that 4-hydroxytamoxifen is a precursor for adduct fraction 3 in vivo. The in vitro adduct fraction 5 (from tamoxifen) was identical to that formed in vivo, indicating that the metabolic pathway for the formation of group II adducts did not involve 4-hydroxytamoxifen. In conclusion, the results support a model where primary metabolites of tamoxifen undergo secondary metabolism to form DNA adducts, which are detected in vivo after treatment with tamoxifen or 4-hydroxytamoxifen.

Animals↗
PubMed1996

DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling.

Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. 32P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with 32P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6 x 10(6) DNA nucleotides in skin (both extracts) and one adduct in 3.2 x 10(7) or 1.2 x 10(7) DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the 32P-labeled derivative of the reaction product of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N2 of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by 32P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts.

Animals↗
PubMed1996

Pentachlorophenol enhances 9-hydroxybenzo [a] pyrene-induced hepatic DNA adduct formation in vivo and inhibits microsomal epoxide hydrolase and glutathione S-transferase activities in vitro: likely inhibition of epoxide detoxication by pentachlorophenol.

We recently reported that co-administration to female mice of tamoxifen or 4-hydroxytamoxifen (4-OH-tamoxifen) with pentachlorophenol (PCP), but not with 2,6-dichloro-4-nitrophenol (DNCP) results in strong intensification of a specific subgroup, termed group I, of tamoxifen-DNA adducts in female mouse liver. As both PCP and DCNP are sulfotransferase inhibitors, we concluded that the intensification of tamoxifen group I adducts is probably not due to inhibition of sulfation by these phenols of a tamoxifen metabolite. Since epoxide derivatives of 4-OH-tamoxifen are potential candidates involved in tamoxifen-induced DNA damage, the hypothesis was developed and tested that PCP inhibits epoxide detoxication. As 4-OH-tamoxifen metabolites were unavailable to us, we employed indirect approaches to test this hypothesis. In the first set of experiments we determined whether PCP would augment DNA adduct formation from the benzo[a]pyrene metabolite, 9-hydroxybenzo[a]pyrene (9-OH-BP), as 9-OH-BP-4,5-epoxide is known to be involved in the metabolic activation of this compound. Female mice were given a single i.p. dose of 9-OH-BP (50 mumol/kg) either alone or in combination with PCP (75 mumol/kg), and hepatic DNA adducts were measured 24 h later by nuclease P1-enhanced bisphosphate 32P-postlabeling. Co-administration of PCP with 9-OH-BP resulted in a statistically significant 1.5- to 1.7-fold increase in 9-OH-BP adduct levels versus 9-OH-BP controls. In order to determine whether PCP inhibits the enzymatic detoxication of epoxides in vitro, in a second set of experiments, the effects of PCP on liver microsomal epoxide hydrolase (mEH) and purified equine liver glutathione S-transferase (GST) activities were studied using, respectively, styrene-7,8-oxide and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. Incubation of mouse liver microsomes with PCP (10-100 microM) strongly inhibited (by 21-97%) mEH activity in a dose-dependent manner, the IC50 being 35 microM. DCNP was ineffective as a mEH inactivator. PCP also inhibited purified equine liver GST activity, with an IC50 of 23.5 microM. Taken together, the results of this study strongly support the hypothesis that PCP inhibited enzymatic detoxication of epoxides in vivo and in vitro. By this mechanism PCP would lead to enhancement of DNA damage caused by 9-OH-BP, and possibly other drugs and their metabolites, which undergo epoxidation prior to DNA binding.

Animals↗
PubMed1996

Organ-specific oxidative DNA damage associated with normal birth in rats.

Mammalian DNA contains bulky endogenous DNA modifications (I-compounds), which increase with age in unexposed animals, as shown by 32P-postlabeling. We have examined the perinatal formation of a subclass (type II) of I-compounds in rat liver, kidney, skin and lung. These I-compounds represent bulky oxidative DNA lesions, defined herein as intrastrand base-base and base-sugar cross-links, adducts of lipid peroxidation products and DNA-protein cross-links. We observed a rapid increase in the levels of five bulky oxidative DNA lesions during the first hours after normal birth of rats, with total levels increasing 4.2-, 3.0- and 1.3-fold, respectively, in liver, kidney and skin. This effect was not noted in lung. The results were consistent with oxidative stress induced by the known sudden increase in partial oxygen pressure at birth in blood and tissues, implying inadequate antioxidant defenses in the affected neonatal organs. Hepatic oxidative damage appeared intensified by increased concentrations of pro-oxidants and reduced concentrations of antioxidants in the maternal diet. The postnatal DNA lesions are postulated to be premutagenic, as indicated by their bulky nature and persistence. Pathophysiological effects of oxidative DNA damage would be exacerbated by rapid cell proliferation in neonatal tissues and consequent fixation as mutations. In addition to inherited mutations, DNA lesions acquired as a consequence of normal birth may play a hitherto unrecognized role in spontaneous carcinogenesis and age-related degenerative diseases.

Animals↗
PubMed1996

DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Animals↗
PubMed1995

Effects of a single dose of the cytochrome P450 inducer, beta-naphthoflavone, on hepatic and renal covalent DNA modifications (I-compounds).

I-compounds are age-dependent covalent DNA modifications, which occur in rodent tissues without known carcinogen exposure. A number of studies from our laboratory indicate that I-compounds may serve as biomarkers of carcinogenesis. Recently, we demonstrated significant lowering of liver I-compound levels in rats that were exposed to different cytochrome P450 inducers. In order to gain further mechanistic insights into the possible relationship between P450 induction and I-compound reduction, female Sprague-Dawley rats were administered a single dose of the CYP1A1 inducer, beta-naphthoflavone (BNF) (80 mg/kg), in corn oil (CO) (2 ml/kg) or CO only (2 ml/kg) as vehicle control. Liver and kidney microsomal P450 contents and P450-related enzyme activities and DNA I-compounds were determined at 4, 24, and 48 h after treatment. Liver and kidney I-compounds were analyzed by nuclease P1-enhanced 32P-postlabeling. DNA synthesis was determined by measuring [3H]methylthymidine incorporation. Liver and kidney microsomal P450 contents were elevated by BNF at 24 and 48 h. Ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) were significantly elevated at all time points, with the former displaying a much higher extent of induction. BNF treatment resulted in significant diminution of the levels of several individual and total I-compounds in liver at 48 h, but few effects were seen at the earlier time-points. Kidney I-compounds were also markedly affected by BNF at 48 h, albeit to a lesser extent than in liver. In both tissues, P450 induction preceded I-compound reduction. Taken together, the results of this investigation demonstrate significant diminution of I-compound levels by a single dose of BNF, a CYP1A1 inducer, in a time-dependent manner, suggesting the participation of a specific biochemical process, possibly involving CYP1A1, in the metabolic regulation of these endogenous DNA adducts.

Animals↗
PubMed1995

Rapid decreases in indigenous covalent DNA modifications (I-compounds) of male Fischer-344 rat liver DNA by diquat treatment.

I-compounds are indigenously appearing covalent DNA modifications that can be detected by 32P-postlabeling assay in tissues of normal animals without known exposure to any carcinogens or toxins. Although these compounds have not been structurally identified, indirect evidence from earlier work suggested the possibility of involvement of molecular fragments derived from lipid peroxides. Diquat is a herbicide that stimulates lipid peroxidation and massive intrahepatic oxidant stress through redox cycling-mediated generation of reactive oxygen species. In the present study, we examined the effects of diquat on hepatic I-compounds of male Fischer-344 rats. Two groups of rats, approximately 14 weeks and 8 weeks old, were given a hepatotoxic dose (0.1 mmol/kg) of diquat or equal volumes of saline, i.p. Two and 6 h later plasma alanine aminotransferase (ALT) activities were measured and hepatic DNA I-compound levels were examined by nuclease P1-enhanced 32P-postlabeling. Elevated ALT activities were observed in some animals in both groups, at both time points, but considerable inter-animal variation was seen. A total of 15-16 I-compound fractions were measured in control and in diquat-treated animals, but no extra spots indicative of treatment-induced adducts were detected. Despite the qualitative similarities, the quantities of individual I-compounds were markedly decreased at 2 h in diquat-treated animals of both age groups. In 14 week old rats the hepatic I-compound contents were decreased at 2 h by 22-59%, which was statistically significant (ANOVA, P < 0.05) for all of the 9 polar I-compound fractions and none of the non-polar fractions. Eleven I-spots from this group showed significant negative linear correlations (P < 0.05) with ALT values. In 8 week old rats treated with diquat a 22-43% depletion in I-compound contents was statistically significant for 4 of the 7 nonpolar and 2 of the 8 polar adduct fractions, but there was no significant correlation of I-compound contents with ALT values at the 2 h time point. By 6 h most of the I-spot levels had returned to normal or above normal values in both groups of animals. While most I-spots from 14 week old rats did not correlate with ALT levels at 6 h, two I-spots displayed positive correlations in the 8 week group. Overall, the susceptibility to diquat-associated DNA alterations appeared to differ with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine Transaminase↗
PubMed1995

DNA adducts induced by lipids and lipid peroxidation products: possible relationships to I-compounds.

A methanol-extractable lipid fraction of oats has been found previously to induce three specific I-compounds (age-dependent covalent DNA modifications) in female rat liver DNA, as detected by the 32P-postlabeling assay. The current report used an in vitro system to explore the possible mechanisms involved in the formation of these DNA derivatives. Ground oats or commercial oatmeal were extracted with methanol, and the extracts were incubated with rat lung DNA in vitro. DNA was recovered and analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay. A number of adducts were induced by the in vitro reaction but none of them was identical by chromatographic analysis to oats-specific I-compounds detected in vivo. Addition of rat liver microsomes and cofactors (NADPH or cumene hydroperoxide) to the in vitro reaction also failed to induce any of the oats-specific I-compounds. Pretreatment of oat lipids with soybean lipoxidase and oxygen enhanced formation of most adducts formed in vitro in a dose- and time-dependent manner. Several of these adducts were related to peroxide derivatives of linoleic acid. Chromatographic evidence suggests that one of the major adducts is derived from 4-hydroxynonenal, a reactive intermediate lipid peroxidation product. This adduct was detectable in liver and kidney DNA of untreated rats and its level increased with age. These results were in line with previous in vivo results, suggesting that the oats-specific I-compounds are presumably formed via an indirect mechanism rather than by direct binding of oats components to DNA.

Aging↗
PubMed1995

Intensification and depletion of specific bulky renal DNA adducts (I-compounds) following exposure of male F344 rats to the renal carcinogen ferric nitrilotriacetate (Fe-NTA).

The effects of the renal carcinogen ferric nitrilotriacetate (Fe-NTA) on kidney DNA of male F344 rats were studied to determine whether bulky DNA oxidation products (putative intrastrand crosslinks) could be detected by 32P-postlabeling in the target organ of carcinogenesis. Rats (10-11 weeks old) were given a single dose of Fe-NTA (15 mg Fe/kg body weight) i.p. at 3:00 pm. After 5 h, renal DNA from Fe-NTA-treated and vehicle control animals was assayed by 32P-postlabeling. Thin-layer chromatography and quantitative analysis of two labeled nucleotide fractions of increasing polarity, L and C, showed that three spots (L1, L2, and C3) were intensified 3.5- to 4.2-fold in treated animals. L1 consisted of subfractions L1a, L1b, and L1c, which could be resolved chromatographically. L1c, L2, and C3 were identical to DNA oxidation products generated by the Fenton reaction in vitro, while L1a and L1b apparently did not arise by this mechanism. DNA damage and toxicity appeared reduced in younger animals and animals treated in the morning, presumably due to differences in antioxidant defenses. Liver and lung (non-target organs) DNA did not exhibit enhanced L1, L2, and C3 spots. In addition to augmenting renal I-compounds, Fe-NTA reduced the levels of three major polar kidney I-compounds (C4, C5, and C6) to 22-53% of control. This reduction did not appear to arise by direct oxidative DNA damage, resembling the previously documented loss of liver I-compounds induced by numerous hepatocarcinogens. Two of these I-compounds (C4 and C5) have been reported to exhibit positive linear correlations with median lifespan of male F344 rats. The pleiotropic response of kidney I-compound levels to Fe-NTA was consistent with different roles of different types (I and II) of I-compounds in Fe-NTA-mediated renal carcinogenesis. The results strongly support a causal relationship between oxidative DNA lesions and Fe-NTA-mediated carcinogenesis.

Age Factors↗
PubMed1995

Evidence from 32P-postlabeling and the use of pentachlorophenol for a novel metabolic activation pathway of diethylstilbestrol and its dimethyl ether in mouse live: likely alpha-hydroxylation of ethyl group(s) followed by sulfate conjugation.

Diethylstilbestrol (DES), a synthetic stilbene estrogen, is a potent development toxin and carcinogen in humans and rodents. A number of 32P-postlabeling studies suggest that genotoxic effects of DES substantially contribute to these biological effects. The mechanisms involved in DES-mediated genotoxicity are not completely understood, however. As reported here, the structural resemblance of tamoxifen to DES led to the hypothesis that DES may be hydroxylated and sulfated at the allylic C2 and/or C5 of the ethyl side chains in analogy to alpha-hydroxylation and sulfation of and DNA adduct formation by tamoxifen. Female ICR mice were administered 500 mumol/kg DES or its dimethyl ether derivative (DiMeDES), either alone or in combination with the sulfotransferase inhibitor pentachlorophenol (PCP) (75 mumol/kg), once daily for 4 days. Liver DNA adducts were measured 24 h after the last dose by dinucleotide/monophosphate 32P-postlabeling. Administration of DES or DiMeDES led to the formation of a unique and novel pattern of several major DNA adducts which were absent in vehicle controls. With minor exceptions the pattern was qualitatively similar for the two compounds, suggesting rapid O-demethylation of DiMeDES to DES in vivo followed by metabolic activation. Adducts formed in vivo did not chromatographically match DES quinone adducts synthesized in vitro. Co-administration of PCP with DES or DiMeDES significantly decreased adduct formation from either compound, by 33-61%. Taken together, these results are consistent with a hitherto unrecognized pathway of metabolic activation and DNA adduct formation by DES involving the putative hydroxylation of the allylic alpha-carbon of the ethyl side chain(s), followed by formation of DNA-reactive sulfuric acid esters. DES is now known to induce DNA damage in vivo by at least four different mechanisms. It is postulated that this multiplicity of mechanisms in itself explains why this drug elicits such a plethora of unique and complex pathophysiological effects in adults and off-spring of different species.

Animals↗
PubMed1994

DNA damage induced by wood preserving waste extracts in vitro without metabolic activation, as assayed by 32P-postlabeling.

Aqueous wood preserving waste (WPW) extracts were tested for their ability to damage DNA in vitro without metabolic activation. Two extracts were prepared from a surface tar and a surface clay soil sample of a WPW site. As assayed by 32P-post-labelling incubation of DNA with these extracts gave rise to highly complex, extract-specific profiles of DNA adducts whose formation depended on the concentration of WPW material. Most of the adducts appeared to be derived from polycyclic aromatic hydrocarbons (PAHs). Three mg organic WPW residue gave rise to total adduct levels of 13.8 (extract 1) and 66.2 (extract 2) DNA modifications in 10(7) DNA nucleotides, corresponding to 13.9 and 26.9 modifications, respectively, per 10 mg of soil. Thus, extract 2 was more active, although the parent residue had a 1.4-times lower PAH content as determined by gas chromatography/mass spectrometry (GC/MS). DNA adduct formation presumably was a consequence of (i) free radical reactions, possibly involving semiquinones and oxygen free radicals, and (ii) reaction of direct-acting electrophiles, derived from metabolism of WPW toxicants by soil microorganisms. These reactions appeared to be more active in sample 2. The results suggest that ground water at WPW sites contains DNA-reactive compounds posing a cancer hazard to humans. The in vitro DNA adduct assay represents a novel tool to readily assess this type of hazard and the possible effects of remediation measures.

Aluminum Silicates↗
PubMed1994

Chemical structure- and time-dependent effects of polycyclic aromatic hydrocarbon-type inducers on rat liver cytochrome P450, DNA adducts, and I-compounds.

It is well documented that cytochrome P450IA1 (CYP1A1) plays an important role in carcinogen activation. CYP1A1/1A2 induction may serve as a biomarker of exposure to environmental toxins. In order to explore a specific role of CYP1A1 in metabolism of I-compounds (age-dependent indigenous DNA modifications), 2-month-old female Sprague-Dawley rats were treated ip with corn oil (2 ml/kg) or with one of several CYP1A1 inducers, i.e., dibenz[a,c]anthracene (DBA) (93 mumol/kg), benzo[a]pyrene (BP) (93 mumol/kg), naphthacene (NAP) (93 mumol/kg), or beta-naphthoflavone (BNF) (140 mumol/kg), once daily for 4 days. Levels of total cytochrome P450 and activities of CYP1A1-associated enzymes, i.e., ethoxycoumarin O-deethylase (ECD) and ethoxyresorufin O-deethylase (EROD), were determined in liver microsomes at 1, 8, or 15 days after the last treatment. DNA adducts and I-compounds were analyzed by nuclease P1-enhanced 32P-postlabeling. DNA synthesis rate was determined by measuring [3H]methylthymidine incorporation into DNA. Each inducer significantly elevated the total P450 level at 1 day. The enzyme levels in BP-, NAP-, and BNF-treated animals gradually returned to control by 8 and 15 days, but elevated levels persisted in DBA-treated rats. Similar trends were observed for ECD and EROD activities. DBA and BP, but not NAP and BNF, gave rise to several measurable DNA adducts, which persisted throughout the period of study. All P450 inducers, irrespective of adduct formation, strongly depleted both nonpolar and polar I-compounds, the effects being most pronounced at 1 and 8 days. These results imply a specific role for CYP1A1 in the regulation of I-compound metabolism, in addition to PAH activation.

Animals↗
PubMed1994

Effect of Ni(II) on tissue hydrogen peroxide content in mice as inferred from glutathione and glutathione disulfide measurements.

Studies on Ni(II)-induced carcinogenesis have suggested that oxidative damage caused by Ni(II) may in part be due to increased tissue H2O2 formation. However, there is lack of evidence in vivo. Because of limitations of available methods for direct measurement of the in vivo rate of H2O2 formation in animals, Ni(II)-induced production of H2O2 was estimated from changes in the rate of glutathione disulfide (GSSG) formation. Male B6C3F1 mice (6-8 wk old) were injected i.p. with 170 mumol NiAc2/kg. Biliary efflux and liver, kidney, and lung levels of glutathione (GSH and GSSG) were determined 0-2 h after treatment. In spite of slight increases in tissue GSSG levels by Ni(II), there was no significant change in the biliary efflux of GSSG. Pretreatment with 50 mg/kg (i.p.) of bis-chloroethyl-nitrosourea (BCNU), an inhibitor of GSSG reductase, did not augment the effects of Ni(II) on GSSG formation significantly. Based on these observations, it was apparent that Ni(II) did not change the concentration of H2O2 significantly in vivo.

Animals↗
PubMed1994

Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite.

Exposure to pentachlorophenol (PCP) strongly intensifies the formation of mouse hepatic DNA adducts elicited by oral administration of tamoxifen (TAM), as previously shown by 32P-postlabeling. To explain this effect, PCP was proposed to interfere with the detoxication by sulfate conjugation of an as yet unidentified hydroxylated proximate TAM metabolite. A comparison of the present and earlier results shows that the hepatic TAM adduct pattern in female ICR mice depended on the route of administration of TAM (120 mumol/kg), with oral administration primarily eliciting formation of more polar adducts (termed group I adducts), while after i.p. administration less polar adducts (group II) predominated over group I adducts by a factor of 17.5. All these adducts were also formed in female Sprague-Dawley rats after i.p. dosing with TAM, but total adduct levels were 3.5- to 5-fold higher than in mice. After four daily i.p. treatments, TAM adducts accumulated in mouse liver DNA in a non-linear fashion. Adduct levels were 30-50 times lower in mouse kidney and lung than in liver. The phenolic metabolite 4-hydroxy TAM (120 mumol/kg) exclusively led to formation of polar (group I) hepatic adducts, and this process was stimulated 8-fold by co-administration of PCP (75 mumol/kg). Co-administration of PCP with the parent compound led to an 11-fold enhancement of group I adduct formation; simultaneously, levels of group II adducts were suppressed 6-fold. Another inhibitor of sulfate conjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had no effect on group I adducts, but it reduced group II adduct formation 2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone (75 mumol/kg) did not significantly affect any major TAM adduct, suggesting that PCP itself was the active compound. Similar to group II TAM adducts, the formation of hepatic safrole-DNA adducts was inhibited in female ICR mice by both sulfotransferase inhibitors, consistent with the proposal that metabolic alpha-hydroxylation of the ethyl group of TAM followed by sulfate conjugation represented a mechanism of TAM activation. On the other hand, the strong intensification of group I adducts by PCP and the lack of this effect by 2,6-dichloro-4-nitrophenol suggested that inhibition of sulfate conjugation may not have been the primary mechanism underlying the intensification of group I adducts formed from TAM or 4-hydroxy TAM. The results presented herein demonstrate conclusively that TAM was activated to DNA-reactive compounds along two distinct pathways which contrasted in their responses to metabolic inhibitors.

Animals↗
PubMed1994

Strong intensification of mouse hepatic tamoxifen DNA adduct formation by pretreatment with the sulfotransferase inhibitor and ubiquitous environmental pollutant pentachlorophenol.

Although negative in assays for mutagenicity, the clinically important antiestrogen tamoxifen induces hepatic DNA adduct formation in mice, rats and hamsters, as indicated by 32P-postlabeling, and is a potent hepatocarcinogen in rats. Both phenolic and alcoholic metabolites of tamoxifen have been reported. As these metabolites are potential candidates for sulfate conjugation, we examined whether the sulfotransferase inhibitor pentachlorophenol, a ubiquitous environmental contaminant, modulates hepatic tamoxifen adduct formation in vivo. Female ICR mice were given tamoxifen (45 mg/kg) daily per os for up to 4 days, with and without i.p. pretreatment with pentachlorophenol (20 mg/kg) 1 h before dosing with tamoxifen. At days 1, 2 and 4, liver DNA was analyzed 5 h after tamoxifen administration by a modified monophosphate version of the 32P-postlabeling assay. At day 4, pentachlorophenol pretreatment led to a large increase (13- to 17-fold) of the levels of four tamoxifen adduct fractions, while two adducts appeared unaffected, resulting in an approximately 7-fold enhancement of overall adduct formation. Significant pentachlorophenol related increases were also observed at day 1 and day 2. The mechanism of this effect has not yet been determined, but may involve the inhibition of sulfation of a tamoxifen metabolite(s) involved in the detoxication of the drug to nonelectrophilic derivatives. It was also apparent that there are two pathways of metabolic activation of tamoxifen, one being sensitive and the other resistant to pentachlorophenol.

Animals↗
PubMed1994

32P-postlabeling of bile components: bulky adduct-like behavior in polyethyleneimine-cellulose thin layer chromatography.

The 32P-postlabeling assay has been used widely in carcinogen-DNA adduct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphorylates the 5'-OH groups of adducted nucleotides in the presence of [gamma-32P]ATP. However, as an exception to this property, PNK has been reported to phosphorylate non-adducted carcinogen metabolites, such as tetrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphorylates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [32P]phosphate from [gamma-32P]ATP to rat bile components or purified bile acids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absence of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100,000-fold less efficient than labeling of 2'-deoxyadenosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, was resistant to phosphorylation. On polyethyleneimine-cellulose TLC maps, 32P-labeled rat bile extract gave an array of non-polar radioactive spots which resembled carcinogen-DNA adducts, while 32P-labeled purified bile acids each gave a single spot. These 32P-labeled products liberated 32Pi upon incubation with prostatic acid phosphatases. Two of the radioactive spots obtained from rat bile were identified as phosphorylated taurocholic and taurodeoxycholic acids by co-chromatography with 32P-labeled standards. These findings demonstrate for the first time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination with bile acids and possibly other bulky/hydrophobic alcohols when analyzing DNA samples by 32P-postlabeling.

Adenosine Triphosphate↗