Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples¹

Abstract

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 μg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 μg/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual’s non-workplace exposure to cadmium, or an individual’s CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYP1A2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYP1A2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, W/C, and C/C were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations.

Introduction

Remarkable inter-individual variations in abundance and activity of some human CYP forms have been reported [1], [2], [3], [4], [5], [6], [7]. For example, in one study [3] using a human liver bank of twenty-one samples (fourteen males, six females, and one unknown), the fold variations (number in parentheses) in catalytic activity found for each CYP were reported as follows: 1A2 (3), 2A6 (21), 2C9 (8), 2C19 (175), 2D6 (18), 2E1 (5), 3A4 (18). It is considered that such variation may be due to exposure to particular classes of drugs and compounds of endogenous and exogenous origin in some individuals [1], [3], [8], [9] and to genetically determined variation in CYP expression, since a number of genetic polymorphisms of human genes have been identified to contribute to variability in the abundance of the corresponding CYP proteins [10], [11], [12], [13], [14]. A recent study on genetic polymorphism reported striking differences in CYP1A1, 1B1, and 2D6 gene polymorphism in Caucasians and Japanese [13], [14].

Among the environmental factors that are important sources of CYP variability among individuals, cigarette smoke exposure has been subject to the most intense research. Results have shown that among the many chemicals in tobacco smoke, polycyclic aromatic hydrocarbons may be inducers of CYP1A2 in liver and CYP1A1 in lung and placenta in tobacco smokers [1], [8], [9]. Cadmium is another common environmental toxin found in low levels in cigarette smoke and in most foods [15], [16], [17], [18], [19], [20], [21], but the possible influence of environmental cadmium exposure on CYP expression and inter-individual variation has not been clarified.

In the non-occupationally exposed population, food and tobacco smoke are known to be major sources of cadmium [15], [16], [19], [22]. Cadmium intake in the range of 9–15 μg/day was estimated for an average Australian consumer, using data from the 1996 Market Basket Survey [21]. Potatoes, wheat, cocoa, and meat constitute 46%, 16%, 12%, and 7%, respectively of total cadmium intake. Crustaceans, liver, peanuts, and vegetables each constitute only 2–3% of cadmium in the diet, providing a further 11% to the total intake. Cadmium of dietary and tobacco smoke origin is known to accumulate in the tissues where CYP enzymes are found, including the placenta, although its accumulation in the proximal tubular cells of the kidney cortex appears to be most extensive [20], [22], [23], [24], [25], [26], [27], [28], which explains why kidney is a target for cadmium toxicity in the non-occupationally exposed population. Cadmium in the kidneys accounts for one-third of the total body cadmium burden, while cadmium in the placenta has been found to increase with age of mothers [27]. Cadmium in lung was found to derive mainly from cigarette smoke and from polluted air [22], [29].

The purpose of this present study was to reveal variations in the expression of selected CYP proteins in liver and kidney that may be attributable to human exposure to environmental cadmium, as reflected by levels of cadmium accumulated in liver and kidney cortex samples. Formulation of this hypothesis developed out of work by ourselves and others [30], [31], [32], [33], [34] showing changes in tissue CYP content associated with cadmium administration to rats and rabbits. Other variables were also taken into consideration in this analysis. These variables were donor age, gender, CYP2A6 genotype, levels of exposure to cigarette smoke, and liver histopathology, some of these being established to be a cause of inter-individual variation in CYP expression [35], [36], [37], [38], [39], [40], [41], [42], [43], [44]. The present paper focuses on the following CYP forms: CYP1A1, 1A2, 1B1, 2A6, 2C9, 2C8/19, 2D6, 3A4, and 3A5. Levels of these CYP forms in liver and kidney samples were determined by Western immunoblotting with a panel of polyclonal antipeptide antibodies possessing high specificity and which have previously been validated for use in studying CYP expression in human liver, lung and placenta tissues [1], [6], [8], [9].