PubMed Health⌕ Search

Biomedical subjects

David H Phillips

Publications and source records attributed to David H Phillips.

49 records · Page 3Linked to original sources
PubMed2003

Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells.

3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites, we developed a panel of Chinese hamster V79MZ-h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 microM 3-NBA, or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the (32)P-postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYP1A2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA.

Acetylation↗
PubMed2003

Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome p450 reductase.

Determining the capability of humans to metabolize the suspected carcinogen 3-nitrobenzanthrone (3-NBA) and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility to this environmental contaminant found in diesel exhaust and ambient air pollution. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-NBA. Using two enrichment procedures of the (32)P-postlabeling method, nuclease P1 digestion and butanol extraction, we found that all hepatic microsomes were competent to activate 3-NBA. DNA adduct patterns with multiple adducts, qualitatively similar to those found recently in vivo in rats, were observed. Additionally one major DNA adduct generated by human microsomes was detected. The role of specific cytochromes p450 (p450) and NADPH:p450 reductase in the human hepatic microsomal samples in 3-NBA activation was investigated by correlating the p450- and NADPH: p450 reductase-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-NBA was attributed to NADPH: p450 reductase. Inhibition of DNA adduct formation in human liver microsomes by alpha-lipoic acid, an inhibitor of NADPH: p450 reductase, supported this finding. Using the purified rabbit enzyme and recombinant human NADPH: p450 reductase expressed in Chinese hamster V79 cells, we confirmed the participation of this enzyme in the formation of 3-NBA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells. The role of individual human recombinant p450s 1A1, 1A2, 1B1, 2A6, 2B6, 2D6, 2C9, 2E1, and 3A4 and of NADPH: p450 reductase in the metabolic activation of 3-NBA, catalyzing DNA adduct formation, was also examined using microsomes of baculovirus-transfected insect cells containing the recombinant enzymes (Supersomes). DNA adducts were observed in all Supersomes preparations, essentially similar to those found with human hepatic microsomes and in human cells. Of all of the recombinant human p450s, p450 2B6 and -2D6 were the most efficient to activate 3-NBA, followed by p450 1A1 and -1A2. These results demonstrate for the first time the potential of human NADPH: p450 reductase and recombinant p450s to contribute to the metabolic activation of 3-NBA by nitroreduction.

Animals↗
PubMed2003

Effects of dexfenfluramine on aristolochic acid nephrotoxicity in a rat model for Chinese-herb nephropathy.

Chinese-herb nephropathy (CHN) is a progressive renal interstitial fibrosis initially reported after concomitant intake of an anorexigen, (dex)fenfluramine, and a Chinese herb ( Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acid (AA). We thus tested the possible enhancing effect of the active enantiomer dexfenfluramine (DXF) on AA nephrotoxicity in a rat model for CHN. Groups of 12 salt-depleted male Wistar rats received daily subcutaneous injections of 7 mg/kg body weight DXF (DXF group), 7 mg/kg body weight AA (AA group), a combination of the same doses of AA and DXF (AA+DXF group), or vehicle (control group) for up to 35 days. Six animals per group were killed on day 10 and the remaining six on day 35. Renal function was evaluated by determining serum creatinine and urinary leucine aminopeptidase activity. Histological evaluation of kidney samples was performed and tubulointerstitial injuries were semiquantified. The DXF group did not differ from controls for any parameter. Similarly elevated serum creatinine levels, decreased leucine aminopeptidase enzymuria, and renal lesions were observed in the AA and the AA+DXF groups after both 10 and 35 days. The formation of specific AA-DNA adducts in liver and renal tissue samples was assessed by the (32)P-postlabelling method. Specific AA-DNA adduct levels were significantly increased in kidney tissues from AA+DXF rats compared with AA rats. These functional and histological data suggest that DXF does not enhance AA nephrotoxicity in a rat model for CHN. Further investigations are needed to clarify the mechanism by which DXF may enhance AA-DNA adduct formation.

Animals↗
PubMed2003

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats.

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation.

Animals↗
PubMed2003

Aristolochic acid nephropathy and the peritoneum: Functional, structural, and molecular studies.

BACKGROUND: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy linked to the exposure to aristolochic acid (AA) and characterized by extensive fibrosis and urothelial atypia. Although the fibrotic process has been documented in extrarenal tissues, the involvement of the peritoneum, as well as the efficacy of peritoneal dialysis in AAN patients, remain uncertain. METHODS: The structure of the peritoneal membrane and the expression of basic fibroblast growth factor (bFGF), collagen type III, endothelial nitric oxide synthase (eNOS), and aquaporin-1 (AQP1) were investigated in peritoneal biopsies from an index AAN patient, four other AAN patients, four regular peritoneal dialysis patients, and two controls. Similar methods were used to investigate a rabbit model of AAN after intraperitoneal exposure to high-dose AA. AA-DNA adducts were screened by 32P-postlabeling analysis. RESULTS: The AAN patients had renal failure, renal fibrosis, and urothelial atypia. The peritoneum of AAN patients had a normal structure, lacked cellular atypia, and, in comparison with regular peritoneal dialysis patients and controls, did not show abnormal regulation of fibrotic and endothelial markers. Furthermore, specific AA-DNA adducts were not identified in the peritoneum of AAN patients. In contrast, AA-DNA adducts were detected in peritoneal and kidney tissues of all exposed rabbits, and one of them developed a malignant mesothelioma. CONCLUSION: These data demonstrate the lack of fibrotic and vascular alterations and the absence of cellular atypia in the peritoneum from AAN patients. Thus, peritoneal dialysis should not be discouraged in these patients. Nevertheless, studies in a rabbit model of high-dose AA exposure may suggest a potential risk of peritoneal malignancy.

Aged↗
PubMed2002

Tumour necrosis factor-alpha mediates tumour promotion via a PKC alpha- and AP-1-dependent pathway.

Tumour necrosis factor-alpha (TNF-alpha) deficient mice (TNF-alpha(-/-) mice) are resistant to skin carcinogenesis. Cellular signalling via the transcription factor complex AP-1 is thought to play a key role in tumour promotion. The induction of a specific subset of AP-1 responsive genes thought to be important for tumour development, namely GM-CSF, MMP-9 and MMP-3, was suppressed in TNF-alpha(-/-) compared to wild-type mouse skin in response to the tumour promotor TPA. The differential induction of these genes correlated with a temporal shift in AP-1 activation and c-Jun expression in TNF-alpha(-/-) compared to wild-type epidermis. The major receptor for TPA-induced signalling in basal keratinocytes, PKC alpha, was also differentially regulated in wild-type compared with TNF-alpha(-/-) epidermis. A marked delay in TPA-induced intracellular translocation and downregulation of PKC alpha was observed in TNF-alpha(-/-) epidermis, which correlated with the deregulated TPA-induced AP-1 activation and c-Jun expression. The frequency of DNA adduct formation and c-Ha-ras mutations was the same in wild-type and TNF-alpha(-/-) epidermis after DMBA treatment, suggesting that TNF-alpha was not involved in tumour initiation. These data suggest that the pro-inflammatory cytokine TNF-alpha is a critical mediator of tumour promotion, acting via a PKC alpha- and AP-1-dependent pathway. This may be one mechanism by which chronic inflammation increases susceptibility to cancer.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2002

Mutagenic fingerprint of ozone in human cells.

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

The proteolytic release of genotoxins from cooked beef.

Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs. Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels. We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis. Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K). Genotoxicity was measured using the alkaline single cell-gel electrophoresis ('Comet') assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019. Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated.

Animals↗
PubMed2002

Mutagens in human breast lipid and milk: the search for environmental agents that initiate breast cancer.

Epidemiological studies indicate the involvement of environmental factors in the etiology of breast cancer, but have not provided clear indications of the nature of the agents responsible. Several environmental carcinogens are known to induce mammary tumors in rodents, and the abundance of adipose tissue in the human breast suggests that the epithelial cells, from which breast tumors commonly arise, could be exposed to lipid-soluble carcinogens sequestered by the adipose tissue. In this report we review our studies in which we have examined human mammary lipid, obtained from elective reduction mammoplasties from healthy donors, and human milk from healthy mothers, for the presence of components with genotoxic activity in several in vitro assays. A significant proportion of lipid extracts induced mutations in bacteria and micronuclei in mammalian cells. They also caused DNA damage, detected as single-strand breaks in the alkaline single-cell gel electrophoresis (comet) assay, in both the MCL-5 cell line and in primary cultures of human mammary epithelial cells. Genotoxic activity was also found in a significant proportion of extracts of human breast milk. Viable cells recovered from milk samples showed evidence of DNA damage and were susceptible to comet formation by genotoxic agents in vitro. Genotoxic activity was found to be less prevalent in milk samples from countries of lower breast cancer incidence (the Far East) compared with that in samples from the UK. The agents responsible for the activity in milk appear to be moderately polar lipophilic compounds and of low molecular weight. Identification of these agents and their sources may hold clues to the origins of breast cancer.

Breast Neoplasms↗
PubMed2002

Associations between carcinogen-DNA damage, glutathione S-transferase genotypes, and risk of lung cancer in the prospective Physicians' Health Cohort Study.

DNA damage from polycyclic aromatic hydrocarbons (PAH) and other aromatic/hydrophobic compounds has been implicated in case-control studies as a risk factor for lung cancer, as have common polymorphisms in the glutathione S-transferase (GST) genes involved in carcinogen detoxification. However, their joint effects have not been evaluated in prospective studies, leaving open questions about predictive value of these biomarkers. In this matched case-control study nested within the prospective Physicians' Health Study, we evaluated whether biomarkers measured in white blood cells (WBC) significantly predicted risk, alone and in combination, after controlling for level of smoking. The biomarkers reported here are aromatic/hydrophobic-DNA adducts and polymorphisms in genes coding for the GSTM1 and GSTP1 enzymes. Our study population was composed of 89 cases of primary lung cancer and 173 controls, matched in a 1:2 ratio on smoking, age and duration of follow up. Adducts were measured in WBC DNA by the nuclease P1-enhanced (32)P-post-labeling method. Genotypes (GSTM1 null versus non-null and GSTP1 Val versus GSTP1 Ile) were determined by genomic amplification and restriction fragment length polymorphism analysis. Among current smokers, adducts were significant predictors of lung cancer risk (after adjusting for GST genotypes, OR = 3.10, 95% CI 1.07, 9.01). The combined GSTM1 null/GSTP1 Val genotype was associated with lung cancer overall and especially among former smokers, before and after adjusting for adducts (OR for former smokers = 4.21, CI 1.08, 16.41; adjusted OR = 4.68, CI 1.17, 18.71). Among cases only, adducts were significantly higher among current or former smokers with the GSTM1 non-null/GSTP1 Ile genotype. The two risk factors (adducts and genotypes) appear to be independent predictors of risk. The findings underscore the complex and important role of biological susceptibility as a determinant of risk from carcinogens found in tobacco smoke and other environmental compounds.

Biomarkers↗
PubMed2002

Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase.

3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.

Air Pollutants↗
PubMed2002

Smoking-related DNA and protein adducts in human tissues.

Tobacco smoking causes not only lung cancer but also cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. Tobacco smoke contains many carcinogens that exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. The same electrophilic species also react with cellular proteins. The effects of smoking are evident by the detection of elevated levels of carcinogen-DNA adducts in many human tissues and of carcinogen-protein adducts in blood. Components of tobacco smoke also induce oxidative DNA damage. Systemic exposure to tobacco-derived carcinogens is demonstrated by the observation of elevated levels of DNA adducts in tissues not directly exposed to tobacco smoke. For many of these tissues there is epidemiological evidence, varying from comprehensive to preliminary, that smoking is a causative factor in cancer of that site. The effects of passive smoking, which also causes lung cancer in non-smokers, is also evident in elevated levels of protein adducts in exposed non-smokers so exposed, relative to non-exposed non-smokers. This paper reviews the literature on smoking-related DNA and protein adducts in human tissues and shows how such studies have provided mechanistic insight into the epidemiological associations between smoking and cancer.

Blood Cells↗