Elsevier

Toxicology

Volume 204, Issues 2–3, 15 November 2004, Pages 161-173
Toxicology

Effects of cigarette smoking and exposure to cadmium and lead on phenotypic variability of hepatic CYP2A6 and renal function biomarkers in men

https://doi.org/10.1016/j.tox.2004.06.022Get rights and content

Abstract

Effects of cigarette smoking and exposure to dietary cadmium (Cd) and lead (Pb) on urinary biomarkers of renal function and phenotypic variability of cytochrome P450 2A6 (CYP2A6) were investigated in a group of 96 healthy Thai men with mean age of 36.7 year (19–57 years). In non-smokers, Cd burden increased with age (r = 0.47, P < 0.001). In current smokers, Cd burden increased with both age (r = 0.45, P = 0.01) and number of cigarettes smoked per day (r = 0.32, P = 0.05). Cd-linked renal tubular dysfunction was seen in both smokers and non-smokers, but Pb-linked glomerular dysfunction was seen in smokers only, possibly due to more recent exposure to high levels of Cd and Pb, as reflected by 30–50% higher serum Cd and Pb levels in smokers than non-smokers (P < 0.05). Exposure to dietary Cd and Pb appeared to be associated with mild tubular dysfunction whereas dietary exposure plus cigarette smoking was associated with tubular plus glomerular dysfunction. Hepatic CYP2A6 activity in non-smokers showed a positive association with Cd burden (adjusted β = 0.38, P = 0.006), but it showed an inverse correlation with Pb (adjusted β = −0.29, P = 0.003), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. In contrast, CYP2A6 activity in current smokers did not correlate with Cd or Pb, but it showed a positive correlation with serum ferritin levels (r = 0.45, P = 0.01). These finding suggest that Pb concentrations in the liver probably were too low to inhibit hepatic synthesis of heme and CYP2A6 and that the concurrent induction of hepatic CYP2A6 and ferritin was probably due to cigarette smoke constituents other than Cd and Pb.

Introduction

Cytochrome P450 2A6 (CYP2A6) is one of the genetically polymorphic enzymes, known to be expressed predominantly in the liver and to be an exclusive catalyst of a conversion of coumarin to 7-hydroxycoumarin (Pelkonen et al., 2000, Oscarson, 2001, Raunio et al., 2001). CYP2A6 metablises also the tobacco alkaloid nicotine to cotinine which is metabolised further by CYP2A6 to 3′-hydroxycotinine (Messina et al., 1997, Nakajima et al., 1996, Benowitz et al., 2003). CYP2A6 is therefore known also as nicotine C-oxidase and its phenotype, reflected by urinary 3′-hydroxycotinine:cotinine ratio, showed a positive correlation with daily cigarette consumption (Benowitz et al., 2003). Individuals with the CYP2A6 gene deficiency were found to be protected from smoking dependence. This has led to the hypothesis that the CYP2A6 genetic polymorphism is a sole determinant of individuals smoking behaviour (Sellers et al., 2003). However, conflicting results have been reported, casting doubt on such role of the CYP2A6 genetic polymorphism (see review Tricker, 2003). No striking differences in nicotine metabolism indices have been seen between Japanese versus American Caucasian male smokers despite their marked differences in the CYP2A6 gene polymorphisms (Domino et al., 2003). Further, our phenotype-genotype analysis has revealed little variation in CYP2A6 phenotype was associated with the CYP2A6 genotypes other than the homozygous null (CYP2A6*4/*4) allele genotype (Ujjin et al., 2002). We hypothesise that CYP2A6 phenotypic variability seen among the majority of healthy individuals is due to their exposure to environmental substances, which have the ability to induce or suppress hepatic CYP2A6 expression and these environmental factors in turn determines nicotine clearance and smoking behaviour. In the present study, we explored the potential influence of ubiquitous environmental pollutants cadmium (Cd) and lead (Pb) on hepatic expression of CYP2A6 together with their well-established renal effects (Madden and Fowler, 2000, Satarug et al., 2000).

Evidence for inducibility of hepatic CYP2A6 comes from studies showing the metals cobalt, and tin, drugs and compounds of diverse structures such as phenobarbital, rifampicin, clofibrate, pyrazole, thioacetamide, griseofulvin increase expression of murine CYP2A5, which is an orthologue of human CYP2A6 (Pelkonen et al., 2000, Oscarson, 2001, Raunio et al., 2001, Donato et al., 2000). Some of these chemicals were indeed found to induce also CYP2A6 expression in primary culture human hepatocytes (Donato et al., 2000). The metals Cd and Pb are absorbed by the body via enteral and pulmonary routes from dietary sources, drinking water, and polluted air (WHO, 1992, Galal-Gorchev, 1993, Jarup et al., 1998). The propensity of the Nicotiana species to concentrate Cd independent of soil-Cd content makes Cd an integral constituent of the tobacco (Elinder et al., 1983). Approximately 10% of the inhaled CdO, generated during the burning of cigarettes, is deposited in lung tissues and another 30–40% is absorbed into systemic blood circulation of smokers. Hence, tobacco is a substantial source of exposure to Cd together with a few thousands more substances (WHO, 1992, Satarug et al., 2004a), but there are few human studies on the effects on specific CYPs of exposure to cigarette constituents other than the polycyclic aromatic hydrocarbon. Cd accumulates in the liver and kidneys while Pb preferentially accumulates in the bone (WHO, 1992, Satarug et al., 2002). Cd in liver and Pb in bone are mobilizable to the kidney, providing an opportunity for nephrotoxicity to occur with no additional exposure. The manifestations of Cd nephrotoxicity include proteinuria, calciuria, aminoaciduria, glycosuria, and tubular necrosis (WHO, 1992, Jarup et al., 1998).

Pb effect on liver drug metabolism was reported in early studies where diminished phenazone elimination rates in men who had been occupationally exposed to Pb and who had evidence of clinical Pb poisoning were reversed after the patients had been treated with EDTA chelation therapy (Fischbein et al., 1977, Meredith et al., 1977). A primary effect of Pb which could be the cause of depressed CYP-mediated phenazone metabolism, was its reduction of heme bioavailability via Pb induction of hepatic expression of the enzyme heme oxygenase (Moore et al., 1987) which degrades heme (Tenhunen et al., 1968) and Pb inhibition of the enzyme δ-aminolaevulinate (ALA) synthase of heme synthesis pathway (Moore et al., 1987). However, although Cd is a potent inducer of heme oxygenase (Alam et al., 1989, Alam et al., 2000, Rosenberg and Kappas, 1991) and an inhibitor of ALA synthase (Fujita, 1997) as with Pb, it also has the ability to induce hepatic and renal CYP2A5 in mice (Bartosiewicz et al., 2001, Urbenjapol et al., 2001, Abu-Bakar et al., 2004). Further evidence for Cd liver effects comes from immuno blotting with antipeptide antibody preparations against various CYPs which revealed correlation between tissue Cd contents and the abundance of certain CYPs in post-mortem liver and kidney cortex samples (Baker et al., 2001, Baker et al., 2002, Baker et al., 2003).

In our previous study, enhanced CYP2A6 activity was detected in healthy men and women who had high Cd burden (Satarug et al., 2004a, Satarug et al., 2004b), reflected by their urinary Cd excretion rate, an indicator of long-term Cd exposure (Lauwerys et al., 1994). However, Pb exposure was not determined for the subjects in the previous study. This raises the possibility of co-exposure to Pb being a confounder in the result observed. In another study, a tendency to have high CYP2A6 activity was seen in a group of men who had serum ferritin of greater than 200 μg/L (Ujjin et al., 2002). The present study was therefore undertaken to clarify the effect of exposure to dietary Cd and Pb and that of cigarette smoking on hepatic CYP2A6 phenotypic variability and on the renal function biomarkers, which included urinary excretion of protein, β2-microglobulin (β2-MG) and N-acetyl-β-d-glucosaminidase (NAG). Other factors considered were urinary zinc excretion, age, and iron store status (Custer et al., 1995).

Section snippets

Chemicals

Coumarin tablets (Venalot®) were purchased from Schaper and Brummer GmbH Co. KG (Salgitter, Germany). Urine metal control samples (Lyphochek®) were from Bio-Rad (Australia) for use as a quality control material for urine metal analysis. ICP multi-element standards were from EM Science, EM Industries, Inc. (Whitehouse Station, NJ, USA). Urine β2-MG, and NAG reagent kits were from Eiken and Shionogi Co.(Tokyo, Japan). Urine protein and serum ferritin reagent kits were purchased from Boehringer

Results

Table 1 shows age, and blood and urine chemistry profiles together with Cd and Pb exposure estimates and CYP2A6 activity for non-smokers, current smokers and ex-smokers, separately. The subjects’ ages ranged from 19 to 57 years and an overall mean age was 36.7 years. Average age and body mass index values were not different between groups. For current smokers, average pack-year and average number of cigarettes consumed per day was 13.6 years and 9.4 cigarettes per day. The corresponding value

Age- and smoking-related increment in Cd burden

A lack of an active biochemical process for Cd elimination coupled with renal reabsorption of Cd from the filtrate predicts age-related increment of Cd accumulation in various tissues and organs, notably liver and kidney (WHO, 1992, Jarup et al., 1998, Satarug et al., 2002). Thus, a positive correlation between subjects’ Cd burden and age is expected. Indeed, such expected correlation was seen in all three groups of non-smokers, current smokers and ex-smokers, irrespective of smoking habits (r

Conclusion

This study reveals opposing effects of Pb (a suppressor) and Cd (an inducer) on hepatic CYP2A6 expression in male non-smokers. A lack of association between Cd or Pb burden on CYP2A6 phenotype in current smokers, who recently were more exposed to Pb than non-smokers was consistent with such opposing effects of Pb and Cd. Exposure to Cd and possibly Pb appear to be environmental determinants of CYP2A6 phenotype in healthy men in the present study. Thus, individual’s Cd and Pb exposure may be

Acknowledgements

We thank all volunteers for their enthusiastic cooperation. We gratefully acknowledge advice and support from Ms Chandhanee Itthipanichpong of Pharmacology Department, Chulalongkorn University and Ms Bussabun Thititummatada of Ban Bangapi School, Klong-gume, Bangkok. EnTox is funded by Queensland Health, the University of Queensland, Queensland University of Technology and Griffith University.

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