ReviewThe effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes
Introduction
The process of drug discovery and development has become increasingly reliant on the use of human-derived test systems to screen drug candidates for their metabolism by, inhibition of, and induction of drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes (Bjornsson et al., 2003). Many of these studies are conducted with human liver microsomes or human hepatocytes either in suspension, as is the case for metabolism studies, or in culture, as is the case for enzyme induction studies.
The expression of CYP enzymes is influenced by endogenous factors, such as genetic polymorphisms and hormone levels, and exogenous factors, such as diet (including nutraceutical chemical use), exposure to drugs (including prescription, over-the-counter and illicit drugs), alcohol consumption, and cigarette smoking. In humans, gender does not influence the expression of cytochrome P450 and other drug-metabolizing enzymes to the extent observed in rats and, to a lesser extent, mice. In adult (sexually mature) rats, CYP2A2, CYP2C11, CYP2C13, and CYP3A2 are all male-specific enzymes (i.e., levels in males are 10 times greater than female levels) due to the pulsatile secretion of growth hormone that is characteristic of male rats. In contrast, CYP2C12 and steroid 5α-reductase are female-specific enzymes (female levels > 10× male levels), whereas CYP2A1 is a female-predominant enzyme (female levels ≈ two to three times the male levels) due to the relatively continuous secretion of growth hormone in female rats Parkinson, 2001, Waxman et al., 1991.
In Japan, the Ministry of Health, Labor and Welfare (MHLW) has recently issued a guidance document recommending that induction studies in rats be conducted in females rather than males because the magnitude of CYP enzyme induction tends to be greater in females (www.nihs.go.jp/drug/DrugDiv-E.html). Our own studies (Sonderfan et al., 1987) illustrate the greater inducibility of CYP2B and CYP3A enzyme activity in mature female rats compared with mature males or immature rats, as shown in Fig. 1, Fig. 2. In this study, immature and mature rats of both genders were treated with 3-methylcholanthrene, phenobarbital, or pregnenolone-16α-carbonitrile (PCN), which are prototypical inducers of CYP1A, CYP2B, and CYP3A enzymes, respectively. As shown in Fig. 1, CYP2B (testosterone 16β-hydroxylase) activity in liver microsomes from phenobarbital-treated rats did not vary much (<2 fold) with age or gender. However, the magnitude of CYP2B induction was considerably greater in mature female rats because these rats had the lowest control (uninduced) activity. The same trend is apparent in Fig. 2, which shows induction of CYP3A (testosterone 6β-hydroxylase) activity by PCN. Again, the absolute rate of testosterone 6β-hydroxylation (nmol/mg microsomal protein/min) did not vary appreciably (<2 fold) with age or gender, but the magnitude of induction (fold increase) was considerably greater in mature female rats than the other groups because of the substantially lower control activity in mature female rats.
The lower CYP3A activity in mature female rats is caused by a decline in CYP3A2 levels after female rats, but not male rats, go through puberty Parkinson, 2001, Waxman et al., 1991. Whereas the levels of CYP3A2 are high in immature rats and become sexually differentiated due to a selective decrease in female rats around the time of puberty, the sexual differentiation of CYP2C11 arises for the opposite reason; the levels of CYP2C11 are low in immature rats and selectively increase around puberty in male rats. In mature rats, male have much higher levels of CYP3A2 and CYP2C11 than do female rats, and this is significant because CYP3A2 and CYP2C11 have broad substrate specificities, which contribute to many cases of sex differences in drug metabolism (Ryan and Levin, 1990). This brings us back to the Japanese MHLW guidance document. Another reason this document recommends that enzyme induction be conducted in female rather than male rats is because test article stability tends to be greater in female rats due to their lower levels of CYP3A2, CYP2C11, and other drug-metabolizing enzymes. A third reason is that the expression of CYP2C11 and CYP3A2 is often suppressed by xenobiotic treatment, which can complicate an assessment of enzyme induction if the substrate chosen to monitor induction is metabolized by both the inducible enzyme (e.g., CYP2B) and CYP2C11 or CYP3A2 (or another suppressible enzyme).
The marked age- and gender-dependent expression of drug-metabolizing enzymes observed in rats and, to a lesser extent, mice have not been observed in other species commonly used in drug testing, such as rabbits, dogs, and cynomolgus monkeys. Nor have they been observed in humans, at least not to an extent that has resulted in individualization of drug dosage because of gender or age. (Although dose may be adjusted for treatment of neonates, infants, and even the elderly, drug dosage does not change depending on whether a patient is pre- or postpubertal as it would if the expression of drug-metabolizing enzymes in humans paralleled that in rats.) Consequently, when it comes to in vitro studies conducted with human-derived material, neither the MHLW in Japan nor the United States Food and Drug Administration (US-FDA) and its regulatory counterpart in Europe advocate the use of one gender over the other or the use of material from donors of a specific age group Bjornsson et al., 2003, Tucker et al., 2001. Nevertheless, purchasing practices by some pharmaceutical scientists show a strong bias for human liver microsomes and hepatocytes from either male or female donors. We have measured CYP activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). We have also examined whether hepatocytes isolated from cirrhotic livers can be used for enzyme induction studies. The aim of this analysis was to evaluate whether the age, gender, or ethnicity of the donor should influence the selection of human liver microsomes and hepatocytes for routine studies of drug metabolism and enzyme induction in vitro.
Section snippets
Sources of liver and donor information
The sources of human liver (and the attendant ethical, confidentiality, and safety issues) and the procedures we use to prepare liver microsomes, isolate and culture hepatocytes, and measure CYP enzyme activities have been described elsewhere LeCluyse et al., 1996a, LeCluyse et al., 1996b, LeCluyse et al., 1999, Madan et al., 1999, Madan et al., 2003, Parkinson et al., 1997, Pearce et al., 1996. All of the data presented below were obtained with human livers that were initially procured for
Cytochrome P450 activity in human liver microsomes
Table 1 gives a summary of the activity of the major CYP enzymes in human liver microsomes, which were measured with substrates known to be reasonably specific for the enzyme under investigation. The rates are expressed as mean ± SD, and it is worth noting that, in all cases, the standard deviations are roughly equal to the means, indicating wide variation in enzymatic rates for each CYP enzyme. The variation is so large that it becomes difficult to develop acceptance criteria for human liver
Acceptance criteria for human liver microsomes
Establishing acceptance criteria for human liver microsomes based on measurements of cytochrome P450 activity would seem like a straightforward procedure. One need simply define the lower and upper limits of the normal range of enzyme activity and apply this normal range to each microsomal sample on a PASS–FAIL basis. However, an examination of the properties of one or two enzymes is sufficient to demonstrate that this approach has several limitations. For example, consider the problems in
Cytochrome P450 and UGT activity in isolated human hepatocytes
Table 2 gives a summary of the activity of CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (testosterone 6β-hydroxylation), and UDP-glucuronosyltransferase (UGT) activity toward 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) in 64 samples of human hepatocytes, which were incubated for up to 4 h in vitro. The rates are expressed as mean ± SD, and it is worth noting that, in all cases, the standard deviations are roughly equal to the means, which
Cytochrome P450 induction in cultured human hepatocytes
We recently reported on the induction of multiple CYP enzymes by prototypical inducers in 62 preparations of cultured human hepatocytes (Madan et al., 2003). After a 2-day adaptation period, primary cultures of human hepatocytes were treated with vehicle [dimethylsulfoxide (DMSO), 0.1%, v/v] or one of three prototypical inducers, namely, β-naphthoflavone (33 μM), phenobarbital (250 μM), and rifampin (20 μM). β-Naphthoflavone activates the arylhydrocarbon (Ah) receptor and thereby induces
Conclusions
Overall, the data summarized in this review consistently point to the same conclusion, namely, that CYP enzyme activity in human liver microsomes and hepatocytes varies considerably from one sample to the next, and there is similarly widespread variation in the inducibility of CYP enzymes in cultured human hepatocytes. This variation is observed in human liver samples from males and females, young, middle-aged and elderly donors, and in livers from Caucasians, African Americans, and Hispanics.
Biography
A personal tribute to Ed Bresnick
I first met Ed Bresnick when I was a graduate student at the University of Guelph in Ontario, Canada. Ed was our invited seminar speaker, and I shall never forget the impact of his talk. To appreciate why, I must point out that, before going to Canada, I graduated from the University of Surrey in England, and I did so in 1977. Where I got my degree is less important than when I got my degree, at least for the purpose of this story. Why? Because in the mid-1970s,
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