Toxicology and Applied Pharmacology 199 (2004) 193–209 www.elsevier.com/locate/ytaap Review The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

Andrew Parkinson,* Daniel R. Mudra, Cory Johnson, Anne Dwyer, and Kathleen M. Carroll

XenoTech, LLC, Lenexa, KS 66219, USA Received 30 September 2003; accepted 16 January 2004 Available online 15 April 2004

Abstract

We have measured cytochrome P450 (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). In human liver microsomes, there were no statistically significant differences ( P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6h- hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with h-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in hepatocytes isolated from cirrhotic liver was comparable to that in normal hepatocytes, which supports the ‘‘healthy hepatocyte, sick environment’’ hypothesis of liver cirrhosis. This review summarizes these findings and discusses their implications for the use of human liver microsomes and hepatocytes for in vitro studies of drug metabolism and enzyme induction, which play a key role in drug development. D 2004 Elsevier Inc. All rights reserved.

Keywords: Liver microsomes; Cytochrome P450; Liver cirrhosis

Introduction ducted with human liver microsomes or human hepatocytes either in suspension, as is the case for metabolism studies, or The process of drug discovery and development has in culture, as is the case for enzyme induction studies. become increasingly reliant on the use of human-derived The expression of CYP enzymes is influenced by en- test systems to screen drug candidates for their metabolism dogenous factors, such as genetic polymorphisms and by, inhibition of, and induction of drug-metabolizing hormone levels, and exogenous factors, such as diet (in- enzymes, especially cytochrome P450 (CYP) enzymes cluding nutraceutical chemical use), exposure to drugs (Bjornsson et al., 2003). Many of these studies are con- (including prescription, over-the-counter and illicit drugs), alcohol consumption, and cigarette smoking. In humans, gender does not influence the expression of cytochrome * Corresponding author. XenoTech, LLC, 16825 West 116th Street, P450 and other drug-metabolizing enzymes to the extent Lenexa, KS 66219. observed in rats and, to a lesser extent, mice. In adult E-mail address: [email protected] (A. Parkinson). (sexually mature) rats, CYP2A2, CYP2C11, CYP2C13,

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.01.010 194 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 and CYP3A2 are all male-specific enzymes (i.e., levels in microsomal protein/min) did not vary appreciably (<2 fold) males are 10 times greater than female levels) due to the with age or gender, but the magnitude of induction (fold pulsatile secretion of growth hormone that is characteristic increase) was considerably greater in mature female rats of male rats. In contrast, CYP2C12 and steroid 5a-reductase than the other groups because of the substantially lower are female-specific enzymes (female levels > 10Â male control activity in mature female rats. levels), whereas CYP2A1 is a female-predominant enzyme The lower CYP3A activity in mature female rats is (female levels c two to three times the male levels) due to caused by a decline in CYP3A2 levels after female rats, the relatively continuous secretion of growth hormone in but not male rats, go through puberty (Parkinson, 2001; female rats (Parkinson, 2001; Waxman et al., 1991). Waxman et al., 1991). Whereas the levels of CYP3A2 are In Japan, the Ministry of Health, Labor and Welfare high in immature rats and become sexually differentiated (MHLW) has recently issued a guidance document recom- due to a selective decrease in female rats around the time of mending that induction studies in rats be conducted in puberty, the sexual differentiation of CYP2C11 arises for the females rather than males because the magnitude of CYP opposite reason; the levels of CYP2C11 are low in imma- enzyme induction tends to be greater in females (www.nihs. ture rats and selectively increase around puberty in male go.jp/drug/DrugDiv-E.html). Our own studies (Sonderfan et rats. In mature rats, male have much higher levels of al., 1987) illustrate the greater inducibility of CYP2B and CYP3A2 and CYP2C11 than do female rats, and this is CYP3A enzyme activity in mature female rats compared significant because CYP3A2 and CYP2C11 have broad with mature males or immature rats, as shown in Figs. 1 and substrate specificities, which contribute to many cases of 2. In this study, immature and mature rats of both genders sex differences in drug metabolism (Ryan and Levin, 1990). were treated with 3-methylcholanthrene, phenobarbital, or This brings us back to the Japanese MHLW guidance pregnenolone-16a-carbonitrile (PCN), which are prototypi- document. Another reason this document recommends that cal inducers of CYP1A, CYP2B, and CYP3A enzymes, enzyme induction be conducted in female rather than male respectively. As shown in Fig. 1, CYP2B (testosterone 16h- rats is because test article stability tends to be greater in hydroxylase) activity in liver microsomes from phenobar- female rats due to their lower levels of CYP3A2, CYP2C11, bital-treated rats did not vary much (<2 fold) with age or and other drug-metabolizing enzymes. A third reason is that gender. However, the magnitude of CYP2B induction was the expression of CYP2C11 and CYP3A2 is often sup- considerably greater in mature female rats because these rats pressed by xenobiotic treatment, which can complicate an had the lowest control (uninduced) activity. The same trend assessment of enzyme induction if the substrate chosen to is apparent in Fig. 2, which shows induction of CYP3A monitor induction is metabolized by both the inducible (testosterone 6h-hydroxylase) activity by PCN. Again, the enzyme (e.g., CYP2B) and CYP2C11 or CYP3A2 (or absolute rate of testosterone 6h-hydroxylation (nmol/mg another suppressible enzyme).

Fig. 1. Effect of age and gender on CYP2B activity and inducibility in rats. (A) Enzyme activity. (B) Fold induction. A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 195

Fig. 2. Effect of age and gender on CYP3A activity and inducibility in rats. (A) Enzyme activity. (B) Fold induction.

The marked age- and gender-dependent expression of Americans, and Hispanics due to a paucity of livers from drug-metabolizing enzymes observed in rats and, to a lesser Asian donors). We have also examined whether hepatocytes extent, mice have not been observed in other species isolated from cirrhotic livers can be used for enzyme commonly used in drug testing, such as rabbits, dogs, and induction studies. The aim of this analysis was to evaluate cynomolgus monkeys. Nor have they been observed in whether the age, gender, or ethnicity of the donor should humans, at least not to an extent that has resulted in influence the selection of human liver microsomes and individualization of drug dosage because of gender or age. hepatocytes for routine studies of drug metabolism and (Although dose may be adjusted for treatment of neonates, enzyme induction in vitro. 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 Sources of liver and donor information humans paralleled that in rats.) Consequently, when it comes to in vitro studies conducted with human-derived The sources of human liver (and the attendant ethical, material, neither the MHLW in Japan nor the United States confidentiality, and safety issues) and the procedures we Food and Drug Administration (US-FDA) and its regulatory use to prepare liver microsomes, isolate and culture counterpart in Europe advocate the use of one gender over hepatocytes, and measure CYP enzyme activities have the other or the use of material from donors of a specific age been described elsewhere (LeCluyse et al., 1996a, 1996b, group (Bjornsson et al., 2003; Tucker et al., 2001). Never- 1999; Madan et al., 1999, 2003; Parkinson et al., 1997; theless, purchasing practices by some pharmaceutical sci- Pearce et al., 1996). All of the data presented below were entists show a strong bias for human liver microsomes and obtained with human livers that were initially procured hepatocytes from either male or female donors. We have for transplantation purposes. All donors were free of measured CYP activity in nearly 150 samples of human infectious diseases that would have precluded transplan- liver microsomes and 64 samples of cryopreserved human tation (such as hepatitis and HIV), and in all cases the hepatocytes, and we have performed induction studies in cause of death was known. All livers were perfused with over 90 preparations of cultured human hepatocytes. We ice-cold organ-preservation solution [usually Belzer’s so- have analyzed these data to examine whether the expression lution, a.k.a. University of Wisconsin (UWI) solution] of CYP enzyme activity in liver microsomes and isolated within a short time of organ procurement, hence, warm hepatocytes or the inducibility of CYP enzymes in cultured ischemia time was kept to a minimum. There are many hepatocytes is influenced by the gender, age, or ethnicity of reasons why livers procured for transplantation are not the donor (the latter being limited to Caucasians, African subsequently transplanted, and these have been reviewed 196 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 elsewhere (Parkinson et al., 1997). Perfusion with cold assess an individual’s overall CYP activity, which would preservation solution (i.e., a short warm-ischemia time) is be the clinically relevant end-point. extremely important both for organ transplantation and The importance of these additional factors is apparent research purposes. To verify this, we processed two livers from studies of CYP enzyme activity in microsomes from that were not perfused with Belzer’s (UWI) solution, and cirrhotic livers. On a per-mg-protein basis, microsomes neither liver yielded microsomes with any cytochrome from cirrhotic livers have CYP activities that tend to be P450 activity (with the interesting exception of CYP4A lower than those in microsomes from histologically nor- activity, which was reduced in these microsomal samples mal livers. Some CYP enzyme activities are decreased but not eliminated, possibly because the heme moiety in less than others; hence, in many liver samples from CYP4A enzymes is covalently attached to the apoprotein; cirrhotic patients, the activity of some CYP enzymes, Hoch and Ortiz De Montellano, 2001). Data for these two including CYP3A4, remain in the normal range of liver atypical livers (the only livers that did not meet our microsomal activity, albeit toward the lower region selection criteria) are not included in the following tables (George et al., 1995a; Guengerich and Turvy, 1991). This and figures. gives the potentially misleading impression that patients The donor information provided with each liver is with liver cirrhosis have only slightly diminished capacity taken at face value. Some of this information, such as for metabolizing drugs at normal rates, which is not the the age and sex of the donor, can be considered highly case. This apparent discrepancy between the in vitro reliable. Other information is incomplete or more difficult finding and in vivo observation is partially resolved by to interpret. For example, information on drug, drinking, taking into account that the amount of microsomal protein and smoking history may be incomplete or misleading. It recovered from cirrhotic liver is less than normal, but is unreasonable to assume that self-proclaimed drinkers even this does not take into account that altered blood and smokers continue drinking alcoholic beverages and flow (due to the formation of portal shunts) and impaired smoking cigarettes during their hospitalization, and it is diffusion of drugs and nutrients (due to the fibrous naive to assume that everyone accurately reports his or material surrounding hepatocytes) both contribute signifi- her drinking and smoking habits, an issue that becomes cantly to the impairment of drug metabolism that is even more problematic with illicit drug use. We will characteristic of liver cirrhosis (discussed later). return to this topic when we discuss the apparent effect (or rather the apparent lack of effect) of alcohol drinking on CYP2E1 levels and of cigarette smoking on 7-ethox- Cytochrome P450 activity in human liver microsomes yresorufin O-dealkylase (CYP1A2) levels. Information on ethnicity assumes that individuals fall into discrete Table 1 gives a summary of the activity of the major groups, even if the donor’s parents are ethnically distinct. CYP enzymes in human liver microsomes, which were This complicates an evaluation of the effects of ethnicity measured with substrates known to be reasonably specific on CYP enzyme expression. On the subject of ethnicity, it for the enzyme under investigation. The rates are ex- is worth mentioning that, for cultural reasons, Asians are pressed as mean F SD, and it is worth noting that, in all less inclined than other ethnic groups to become organ cases, the standard deviations are roughly equal to the donors, hence, this ethnic group is not represented in the means, indicating wide variation in enzymatic rates for following tables and figures. each CYP enzyme. The variation is so large that it In analyzing our data, we have not applied selection becomes difficult to develop acceptance criteria for human criteria when evaluating the effects of any given variable liver microsomes (especially based on a single enzyme on CYP enzyme activity. For example, when evaluating activity), which raises the question: How do we know our the effects of gender, we used all the available data. We samples of human liver microsomes are intact and contain did not exclude samples from donors who, for example, normal levels of cytochrome P450? Before analyzing were reportedly taking enzyme-inducing drugs. That is because our goal was to assess whether age, gender, and Table 1 ethnicity should be taken into account when selecting Enzyme Activity Rate n human liver microsomes or human hepatocytes for in CYP1A2 7-ethoxyresorufin O-dealkylation 49.9 F 38.0 142 vitro drug metabolism or enzyme induction studies, rather CYP2A6 coumarin 7-hydroxylation 939 F 990 139 than to discern whether any of these variables have CYP2B6 S-mephenytoin N-demethylation 122 F 178 135 clinically relevant influences on drug metabolism. To CYP2C8 paclitaxel 6a-hydroxylation 348 F 340 122 attempt the latter (i.e., to extrapolate from the in vitro CYP2C9 diclofenac 4V-hydroxylation 1930 F 840 129 V F to the in vivo situation) would require a much larger CYP2C19 S-mephenytoin 4 -hydroxylation 108 142 142 CYP2D6 dextromethorphan O-demethylation 299 F 202 141 sample size than we possess (so that selection and CYP2E1 chlorzoxazone 6-hydroxylation 1950 F 980 142 exclusion criteria could be applied), and it would require CYP3A4 testosterone 6h-hydroxylation 3630 F 3470 142 additional information (such as the amount of microsomal CYP4A11 lauric acid 12-hydroxylation 1970 F 850 142 protein per gram of liver and the weight of the liver) to Note. Rates are reported in pmol/mg protein/min. A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 197 whether age, gender, and ethnicity have an effect on the CYP2D6 activity in liver microsomes from a homozy- microsomal cytochrome P450 activity, it is pertinent to gous positive (+/+) individual would represent the upper address this question. limit of normal for CYP2D6 activity, but this is not the case. Some individuals (particularly those from the Mediterranean area, Spain, and certain regions of Asia) have multiple Acceptance criteria for human liver microsomes copies of the CYP2D6 gene. The record to date is set by a Chinese individual with 13 functional CYP2D6 genes Establishing acceptance criteria for human liver micro- (Johansson et al., 1993). This individual’s liver is not in somes based on measurements of cytochrome P450 ac- XenoTech’s liver bank but, if it were, it would be considered tivity would seem like a straightforward procedure. One acceptable based on our selection criteria. Therefore, the need simply define the lower and upper limits of the normal range for liver microsomal CYP2D6 activity ranges normal range of enzyme activity and apply this normal from zero to whatever happens to be the current record for range to each microsomal sample on a PASS–FAIL basis. CYP2D6 activity in liver microsomes, which reflects the However, an examination of the properties of one or two record for CYP2D6 gene duplication. Needless to say, ALL enzymes is sufficient to demonstrate that this approach microsomal samples fall within this range of CYP2D6 has several limitations. For example, consider the prob- activities, hence, no microsomal sample is disqualified lems in defining a normal range for CYP2D6, starting solely because of its CYP2D6 activity. For similar reasons, with the lower limit. this is largely true of all of the other individual CYP About 7% of Caucasians are devoid of CYP2D6 activity enzymes in human liver microsomes. (i.e., they possess two inactive CYP2D6 alleles and conse- Consider now an end-point that shows considerably less quently synthesize no active enzyme at all). (These individ- variation than CYP2D6 activity, and where a zero value uals are called homozygous negative, which is designated would be automatic cause for concern. The end-point À/À.) Therefore, the lower limit of the normal range of displaying the least variation from one microsomal sample CYP2D6 activity in human liver microsomes is zero. It must to the next is cytochrome b5 (technically speaking, this is be emphasized that liver microsomes from CYP2D6-defi- not a drug-metabolizing enzyme, but we routinely measure cient individuals possess ‘‘normal’’ levels of all other CYP cytochrome b5 levels by spectroscopy when we measure the enzymes. In fact, the selective loss of CYP2D6 makes liver levels of cytochrome P450). This would seem to be a good microsomes from À/À individuals (so-called 2D6 PMs, end-point with which to judge the quality of liver micro- which stands for poor metabolizers) useful in some studies. somes because the amount of cytochrome b5 in liver micro- The upper limit of the normal range of CYP2D6 activity somes is not known to be markedly influenced by genetic is difficult to define. To a certain extent, there is no upper factors (in contrast to CYP2D6, for example) or environ- limit to the normal range of CYP2D6 activity. How is this mental factors (as for CYP3A4 and several other CYP possible? Most individuals have two copies (alleles) of a enzymes). single CYP2D6 gene. If only one of the alleles produces Fig. 3 shows the relatively narrow variation in cyto- active enzyme, the individual is heterozygous (designated chrome b5 levels vs. the relatively large variation in +/À) and his or her liver microsomes will contain relatively CYP3A4 activity in our bank of liver microsomes. Similar low (but not zero) CYP2D6 activity. If both alleles produces data on a limited number of samples have been published active enzyme, the individual is homozygous positive (des- previously (Pearce et al., 1996). These data, in contrast to ignated +/+) and his or her liver microsomes will contain data on individual CYP enzyme activity, are useful in relatively high CYP2D6 activity. It would seem logical that helping to accept or reject samples of human liver micro-

Fig. 3. Examples of the range of CYP activities in human liver microsomes. 198 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 somes. The lower limit of the normal range of cytochrome at XenoTech. For example, we do not accept livers from b5 values is about 0.24 nmol/mg protein, and the upper limit donors with a variety of active infectious diseases, which appears to be around 0.9 nmol/mg protein. The sample with would exclude many of the livers with suppressed CYP3A4 the lowest level of cytochrome b5 (0.24 nmol/mg protein) levels. However, we do not exclude livers from donors was judged ‘‘normal’’ because it was not uniformly low in treated with drugs known to increase CYP3A4 levels, which all end-points measured. Compared with the other micro- include many of the anticonvulsant and anti-inflammatory somal samples analyzed, this sample (which corresponds to drugs administered to head trauma patients. Sample 10 in the publication by Pearce et al. (1996)) had It is apparent from Fig. 3 that CYP3A4 activity varies lower-than-average CYP1A2, 2A6, 2C19, 3A4, and 4A11 enormously (> 40 fold) from one microsomal sample to the activity, but it had higher-than-average CYP2C9 activity, in next. Several samples catalyze rates of testosterone 6h- addition to average or middle-of-the-road CYP2D6 and hydroxylation below 0.5 nmol/mg protein/min, and several CYP2E1 activity. The liver microsomes we prepared from others catalyze rates greater than 10 nmol/mg/protein. Those the two human livers that were not perfused with Belzer’s samples with low or high CYP3A4 activity are not uni- (UWI) solution both had cytochrome b5 levels below 0.24 formly low and high, respectively, in all other CYP activ- nmol/mg protein (in addition to having little or no CYP ities, although as a rule, the levels of CYP3A4 are loosely activity). correlated with the levels of CYP2B6 and the CYP2C In terms of a normal range of activity, CYP3A4 falls enzymes presumably because these enzymes are all regu- between cytochrome b5 (which varies about 4-fold from lated, at least in part, by some of the same medications (a high to low) and CYP2D6 activity (which varies infinitely point we will return to in the section on enzyme induction). because zero CYP2D6 activity can be considered normal if From the foregoing, it is probably apparent that we look at the donor possessed two inactive CYP2D6 alleles). An all the end-points measured to qualify or disqualify a given individual with two deficient CYP3A4 alleles (i.e., a true sample of human liver microsomes. A sample with uni- CYP3A4 poor metabolizer) has yet to be described in the formly low or undetectable CYP activity would be disquali- literature, so a microsomal sample devoid of any CYP3A4 fied (as was the case with the two samples prepared from activity would be so unusual that zero CYP3A4 activity non-perfused livers). would likely be ascribed to degradation of the liver sample In the tables of results and figures, the number of (as was the case with the two un-perfused livers). Whereas samples (n) varies slightly from one CYP activity to the CYP2D6 activity varies enormously due to genetic factors, next. This is not because selected data points were CYP3A4 activity varies enormously due to environmental omitted, but because certain samples were not analyzed ones. Many medications are known to increase CYP3A4 for that particular CYP activity or, if they were, another levels, and many disease states (such as viral and bacterial marker substrate was used. infections) are known to suppress CYP3A4 levels (Parkin- son, 2001). Consequently, when it comes to defining ‘‘nor- Effects of gender on cytochrome P450 activity in human mal’’ CYP3A4 activity, one is forced to contend with the liver microsomes intractable issue of defining what constitutes a normal environment. As part of our preselection criteria, we define The effects of gender on CYP3A4 activity in human liver certain abnormal states among our criteria for disqualifying microsomes are shown in Fig. 4 and for all CYP enzymes in human livers, and this probably introduces a certain bias Fig. 5. From the size of the error bars in Fig. 5, and from the into the level of CYP3A4 in the liver microsomes prepared spread of data in Fig. 4, it is apparent that, without

Fig. 4. Effects of gender on the range of CYP3A4 activity in human liver microsomes. A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 199

Fig. 5. Effects of gender on CYP activity in human liver microsomes. No statistically significant differences were determined by linear regression analysis with the exception of CYP1A2, P = 0.03. exception, CYP activity varies widely in liver microsomes Although not statistically significant, the observation that from both male and female donors. The differences in liver microsomal CYP3A4 is slightly greater in females than means between males and females are slight (< 25%) and males is interesting in light of clinical findings. Many clinical not statistically significant with one exception (CYP1A2, studies suggest females have greater CYP3A4 activity than P = 0.03). Despite limited statistical significance, there are males, based on pharmacokinetic studies of cyclosporin, some trends. For example, CYP2B6, CYP2C19, and erythromycin, methylprednisolone, midazolam, nifedipine, CYP3A4 activity tends to be higher in female liver micro- and tirilazad (Meibohm et al., 2002; Tanaka, 1999). However, somes, whereas CYP1A2 and CYP2D6 activity tend to be studies with some of these same drugs (cyclosporin and higher in male liver microsomes. midazolam) and studies with alprazolam and alfentanil sug- The clinical effects of gender on drug disposition have gest there is no gender difference in CYP3A4 activity. been reviewed by Beierle et al. (1999), Cummins et al. Interpretation of clinical reports of gender differences in the (2002), Harris et al. (1995), Meibohm et al. (2002) and CYP3A4-depedendent metabolism of drugs is complicated Tanaka (1999). Clinical studies have shown that, in Cauca- by the possibility that increased metabolism of drugs by sian, African American, and Asian (Chinese) populations, CYP3A4 in women relative to men may actually reflect woman have lower CYP1A2 activity than men based on lower P-glycoprotein activity in the hepatic canalicular mem- pharmacokinetic studies of caffeine, thiothixene, naproxen, brane, rather than greater CYP3A4 activity in the endoplas- and clozapine (all of which are substrates for CYP1A2). mic reticulum (Cummins et al., 2002). Schuetz et al. (1995) However, not all clinical studies support this finding, and in reported that P-glycoprotein levels vary widely in normal fact, at least one report (based on a study of theophylline liver samples, but on average, women have about half the P- metabolism) suggests that females actually have higher glycoprotein levels of men. The lower expression of P- CYP1A2 activity than males (Nafziger and Bertino, 1989). glycoprotein in women compared with men could result in Rasmussen et al. (2002) recently reported that, based on a higher intracellular concentrations of drug and consequently clinical study of caffeine metabolism in 378 individuals, greater metabolite formation. The reason this does not result males have greater CYP1A2 activity than females. Although in a gender difference in the metabolism of drugs by all CYP oral contraceptives reduce CYP1A2 activity, the gender enzymes is because most substrates for P-glycoprotein are difference reported by Rasmussen et al. was significant also substrates for CYP3A4 (Cummins et al., 2002; Wacher et even when women on oral contraceptive steroids were al., 1995). This issue will be discussed further when we later excluded from the analysis. Rasmussen et al. (2002) also examine CYP3A4 activity in intact hepatocytes. demonstrated a strong correlation in CYP1A2 activity A final comment on the data in Fig. 5 will serve to provide between identical (monozygous) twins and concluded that yet another example of the difficulty in extrapolating in vitro genetic factors account for 70–75% of the observed varia- results to the in vivo situation. Although the difference is tion in CYP1A2 activity. Although our finding that small and not statistically significant, the data in Fig. 5 CYP1A2 activity is significantly greater in liver microsomes suggest that, if anything, females have higher levels of from males than females is largely consistent with clinical CYP2C19 compared with males. However, there is a clinical observation, the gender difference in vitro activity is rela- report, based on the metabolism of (R)-mephobarbital, that tively small (<25%). suggests young women (but not elderly women) have lower 200 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209

CYP2C19 activity compared with males (Hooper and Qing, are analyzed. However, given the range of activities for each 1990), which contrasts with several other clinical studies CYP enzyme in each age group, it seems doubtful that such showing no gender difference in CYP2C19 activity (Mei- trends will readily reach statistical significance. bohm et al., 2002). Subsequent studies revealed that the The clearance (and volume of distribution) of numerous apparent gender difference observed with (R)-mephobarbital drugs is diminished in elderly people, although this seems to can be attributed to concomitant use of oral contraceptive reflect a decrease in liver volume and hepatic blood flow steroids, which reduces CYP2C19 activity by about two- (Marchesini et al., 1988; Wynne et al., 1989), rather than a thirds. Oral contraceptive use not only explains the apparent decrease in liver microsomal enzyme activity (Schmucker et gender difference in (R)-mephobarbital metabolism, but also al., 1990). Our findings (Fig. 6) are consistent with this explains why this apparent gender difference was restricted to interpretation because there is little difference in liver micro- young women (Meibohm et al., 2002). somal CYP activity between adult (20–60 years old) and elderly people (> 60 years old). The fact that diminished liver Effects of age on cytochrome P450 activity in human liver volume and diminished or altered blood flow are clinically microsomes important causes of the diminished capacity for drug metab- olism seen in elderly people and patients with cirrhosis (or The effects of age on CYP enzyme activity in human liver other liver disease) underscores the caution that must be microsomes are shown in Fig. 6. The data were arranged into exercised when extrapolating in vitro data to the in vivo three age groups: less than 20 years old, between 20 and 60 situation. years old, and older than 60 years old. For each CYP enzyme, there were at least 19 samples in each of these three age Effects of ethnicity on cytochrome P450 activity in human groups. Although further subdivision was desirable, this liver microsomes created groups with unacceptably small numbers. From the size of the error bars in Fig. 6, it is apparent that, without The effects of ethnicity on CYP enzyme activity in exception, CYP activity varies widely in liver microsomes human liver microsomes are shown in Fig. 7. For some from donors of all ages, and there are few statistically CYP enzymes, data for Hispanics are available for only 10 significant differences for CYP enzymes among the three microsomal samples, so these data should be interpreted age groups (<20 years, 20–60 years, and >60 years old). with particular care. From the size of the error bars in Fig. 7, Although only CYP1A2 ( P = 0.018), CYP2D6 ( P = 0.015), it is apparent that, without exception, CYP activity varies and CYP2E1 ( P = 0.006) were found to be significantly widely in liver microsomes from donors representing three different, some age-related differences in mean values were ethnic groups, and there are no statistically significant observed. For example, CYP1A2, CYP2B6, CYP2C19, differences in CYP activity among Caucasians, African CYP2D6, and CYP2E1 activity appeared to decrease (at least Americans, and Hispanics. Compared with those from 25%) with age, and CYP2A6 and CYP4A11 activity Caucasians and African Americans, liver microsomes from appeared to increase (at least 25%) with age. It remains to Hispanics tended to have two to three times greater be seen whether these trends will persist as additional samples CYP2A6, CYP2B6, and CYP2C8 activity and about half

Fig. 6. Effects of age on CYP activity in human liver microsomes. No statistically significant differences were determined by linear regression analysis with the exception of CYP1A2, P = 0.018; CYP2D6, P = 0.015; and CYP2E1, P = 0.006. A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 201

Fig. 7. Effects of ethnicity on CYP activity in human liver microsomes. No statistically significant differences were determined by linear regression analysis.

the CYP1A2 activity, although this apparent ethnic differ- smoke cigarettes during their hospitalization. Furthermore, ence may be a consequence of the relatively low number of our analysis does not exclude other factors, either genetic Hispanic donors. It will be interesting to see if these or environmental, that contribute substantially to variation apparent differences are maintained as additional data on in CYP1A2 levels. In the case of genetic factors, Ras- Hispanic samples become available, and whether such mussen et al. (2002) identified a strong correlation in differences have corresponding clinical significance. CYP1A2 activity between identical (monozygous) twins and suggested that genetic factors account for approxi- Effects of cigarette smoking on CYP1A2 activity in human mately 75% of the observed variation in CYP1A2 activ- liver microsomes ity. In the case of environmental factors, certain drugs (e.g., omeprazole), cruciferous vegetable (e.g., broccoli), Cigarette smoking is known to cause clinically signifi- strenuous exercise, and infection diseases (including vac- cant induction of liver microsomal CYP1A2. For example, cinations) are all known to influence CYP1A2 expression, Rasmussen et al. (2002) reported that the N-demethylation as does stage of menstrual cycle (Vistisen et al., 1991). of caffeine to paraxanthine, a clinically reliable indicator of To a certain extent, the influence of genetic and environ- CYP1A2 activity, is greater in males than females, but is mental factors on the results of clinical studies is reduced inducible in both genders by cigarette smoking. Fig. 8 shows by taking measurements before and after a particular liver microsomal CYP1A2 activity as a function of gender and cigarette smoking. This analysis was restricted to those samples for which we received unambiguous data on the donor’s smoking habits. Fig. 8 shows that male nonsmokers had a greater range of CYP1A2 activity than the corresponding female group, consistent with the clinical finding of a gender difference (males > females) in CYP1A2 activity. In females, the samples with the highest CYP1A2 were from smokers, but this was not the case for male donors. The in vitro data in Fig. 8 offer few clues to the clinical observation that cigarette smoking induces CYP1A2 activity in vivo. Despite our efforts to use only those data for which we had clear-cut data on smoking habits, it is possible that the information available to us is incomplete or inaccu- rate. For example, some smokers may have identified themselves (or been identified by next of kin) as being nonsmokers. Even when smokers are correctly identified, it is unreasonable to assume that all these patients (some Fig. 8. Apparent effects of smoking cigarettes on CYP1A2 activity in of whom may be on life-support systems) will continue to human liver microsomes. 202 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209

analysis does not exclude other factors, either genetic or environmental, that contribute substantially to variation in CYP2E1 levels. The data in Fig. 9 do not control for such factors. From a practical consideration, it might be assumed that choosing microsomal samples from known alcohol drinkers would be helpful in selecting those samples with high CYP2E1 activity, but the results in Fig. 9 argue against this assumption.

Cytochrome P450 and UGT activity in isolated human hepatocytes

Table 2 gives a summary of the activity of CYP2D6 (dextromethorphan O-demethylation), CYP2E1 (chlorzoxa- Fig. 9. Apparent effects of drinking alcohol on CYP2E1 activity in human zone 6-hydroxylation), CYP3A4 (testosterone 6h-hydroxyl- liver microsomes. ation), and UDP-glucuronosyltransferase (UGT) activity toward 4-methylumbelliferone (7-hydroxy-4-methylcou- marin) in 64 samples of human hepatocytes, which were treatment, in which case each person serves as his or her incubated for up to 4 h in vitro. The rates are expressed as own control. This in vivo approach is not applicable to in mean F SD, and it is worth noting that, in all cases, the vitro systems like liver microsomes. From a practical standard deviations are roughly equal to the means, which consideration, it might be assumed that choosing micro- reflects the wide variation in enzymatic rates for each somal samples from known cigarette smokers would be enzyme. helpful in selecting those samples with high CYP1A2 As for microsomes, the large variation in enzyme activity, however, the results in Fig. 8 argue against this activity raises questions about the test system itself. In assumption. contrast to microsomes, the normalcy and acceptability of hepatocytes can be based, at least in part, on cell Effects of alcohol drinking on CYP2E1 activity in human morphology and viability. The data presented in Table liver microsomes 2 and in Figs. 10 and 11 are from human hepatocytes that were viable and remained so during the incubation Alcohol consumption is known to cause clinically (which continued for at least 2 h and up to 4 h, during significant induction of liver microsomal CYP2E1. For which time multiple measurements of cell viability and example, Girre et al. (1994) reported that the 6-hydroxyl- metabolite formation were made). As for microsomes, ation of chlorzoxazone, a clinically reliable indicator of acceptability of hepatocytes is based, at least in part, on CYP2E1 activity, is inducible by alcohol consumption. the observation that not all enzyme activities are uni- The results in Fig. 9 show CYP2E1 activity in liver formly low in a given sample. In the case of CYP2D6, microsomes as a function of gender and alcohol consump- we have also established that the variation in enzyme tion. This analysis was restricted to those samples for activity in isolated hepatocytes is highly correlated with which we received unambiguous data on the donor’s CYP2D6 activity in microsomes prepared from the same drinking habits. Fig. 9 shows that there were no meaning- liver (Mudra et al., 2001). ful differences between males or females or between drinkers and nondrinkers. The arguments offered to account for the apparent lack Table 2 of association between cigarette smoking and microsomal Enzyme Activity Rate n CYP1A2 activity also apply to the apparent lack of F association between alcohol consumption and microsomal CYP2D6 dextromethorphan 300 200 64 O-demethylation CYP2E1 activity. In other words, drinking habits may not CYP2E1 chlorzoxazone 2060 F 1060 64 have been accurately reported, and it is unreasonable to 6-hydroxylation assume that all patients correctly identified as drinkers CYP2B6/ dextromethorphan 81.9 F 79.6 63 continued to consume alcohol during their hospitalization. CYP3A4/5 N-demethylation F Furthermore, although cigarette smokers tend to smoke a CYP3A4/5 testosterone 3910 3370 64 6h-hydroxylation certain number of cigarettes per day, some self-proclaimed UGT 4-methylumbelliferone 1990 F 900 63 drinkers consume alcohol sporadically, perhaps only on glucuronidation the weekend, hence, the time between alcohol consump- Note. Rates are reported in pmol/mg protein/min except for UGT, which is tion and liver procurement can vary enormously. Our in nmol/mg protein/min. A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 203

Fig. 10. Effects of gender on CYP activity in cryopreserved human hepatocytes. No statistically significant differences were determined by linear regression analysis with the exception of CYP3A4, P < 0.001.

Effects of gender on cytochrome P450 activity in human ylation of testosterone, and testosterone is not a substrate hepatocytes for P-glycoprotein (or, if it is, it is a poor substrate). Accordingly, P-glycoprotein would not be expected to The effects of gender on CYP2D6, CYP2E1, CYP3A4, affect CYP3A4 activity toward testosterone. However, and UGT activity in human hepatocytes are shown in Fig. Baron et al. (2001) reported that testosterone 6h-hydrox- 10. From the size of the error bars, it is apparent that, ylation in a cell line expressing CYP3A4 was influenced without exception, these enzyme activities vary widely in by co-expression of P-glycoprotein, which increased the hepatocytes from both male and female donors. There is apparent Km by 1.7-fold (which implies P-glycoprotein no significant difference between males and females in decreased intracellular levels of testosterone). The effect terms of CYP2D6 and CYP2E1 activity, but there is a was even greater for cortisol (Km increased 4-fold), which significant difference in CYP3A4 activity, with females is a P-glycoprotein substrate. On the other hand, to exhibiting about twice the activity of males. This is larger measure CYP3A4 activity, we incubated hepatocytes with than the difference in CYP3A4 activity seen in liver 250 AM testosterone, which is well above Km, hence, microsomes, which was not statistically significant (Fig. small changes in apparent Km would not be expected to 5). If analysis of a larger number of samples supports influence rates of testosterone 6h-hydroxylation measured these findings, we will need to explain why there is a 2- under conditions of zero-order kinetics. Furthermore, a fold gender difference in CYP3A4 activity in hepatocytes recent paper by Wolbold et al. (2003) argues against a but not in liver microsomes. The explanation might gender difference in P-glycoprotein and argues in favor of involve P-glycoprotein, which could conceivably influ- a gender difference in CYP3A4 as the underlying basis ence CYP3A4 activity in hepatocytes but not liver micro- for the clinical observation of gender-dependent differ- somes. The levels of P-glycoprotein vary widely from one ences in drug clearance. liver sample to the next, but average levels are about In summary, arguments can be made both for and against twice as high in liver samples from males compared with the possibility that the 2-fold gender difference in CYP3A4 females (Schuetz et al., 1995). We have not measured P- activity that we observed in human hepatocytes (female = glycoprotein levels or functional activity in our 64 sam- twice male) reflects a 2-fold gender difference in P-glyco- ples of hepatocytes, so we do not know whether they protein levels (female = half male), and a resolution will show the same gender difference reported by Schuetz et require additional studies. Regardless of the outcome, the al. (1995). If they did, could this explain the observed fact remains that the real or apparent gender difference in gender difference in CYP3A4 activity in hepatocytes? In CYP3A activity in humans is about 2-fold, which is small, other words, is it possible that lower P-glycoprotein especially compared with the marked sex difference (>10 activity in female hepatocytes leads to an increase the fold) observed in rats. Furthermore, within each gender, intracellular level of testosterone, which then increases the CYP3A4 activity varies enormously due, at least in part, to rate of testosterone 6h-hydroxylation, thereby giving the the many environmental factors that induce or suppress impression of greater CYP3A4 activity in female hepato- CYP3A4 (and P-glycoprotein) expression. Such factors, cytes compared with male hepatocytes? The immediate which are the basis of numerous drug–drug interactions, answer would appear to be NO, because we measured have proven far more clinically significant than gender CYP3A4 activity in hepatocytes based on the 6h-hydrox- differences in CYP3A4 (or P-glycoprotein) activity. 204 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209

Fig. 11. Effects of age on CYP activity in cryopreserved human hepatocytes. No statistically significant differences were determined by linear regression analysis.

Effects of age on cytochrome P450 activity in human CYP2C19, and CYP3A4, albeit it to different extents hepatocytes (Parkinson, 2001). Twenty-four hours after the last treat- ment, microsomes were prepared from the cultured hep- The effects of age on CYP2D6, CYP2E1, CYP3A4, and atocytes and analyzed for CYP enzyme activity. UGT activity in human hepatocytes are shown in Fig. 11. In the case of induction studies, acceptability and From the size of the error bars, it is apparent that, without normalcy of cultured hepatocytes can be determined in exception, these enzyme activities vary widely in hepato- large part by the ability of the cells to attach to collagen, to cytes from young (<20 years), middle aged (20–60 years), form cell–cell communications, to assume normal hepato- and elderly donors (>60 years). There is no significant cellular morphology, and to remain attached and viable for difference among these three age groups in any of the several days (even weeks) in culture. In cell cultures that enzyme activities measured. CYP3A4 activity appeared to meet all these requirements, there is still considerable increase with age, a trend that was not apparent in liver variation in the magnitude of CYP induction from one microsomes. The number of samples from Hispanics and culture to the next. There appears to an upper limit to the African Americans was insufficient to determine whether amount of CYP enzyme that cultured hepatocytes can ethnicity of the donor affected enzyme activity in isolated synthesize, and this in vitro upper limit appears to be the hepatocytes. same as that observed in vivo. For example, microsomes Overall, the data summarized in Figs. 10 and 11 suggest from rifampin-treated hepatocytes catalyze rates of testos- that hepatocytes intended for routine in vitro studies of drug terone 6h-hydroxylation that are usually in the range of 6– metabolism and enzyme induction need not be selected 12 nmol/mg protein/min, and occasionally reach 15 nmol/ because of the age or gender of the donor. This is not mg protein/min. From Fig. 3, it is apparent that these in altogether unexpected in view of the corresponding results vitro rates are toward the high end of the range of obtained with liver microsomes. testosterone 6h-hydroxylation rates catalyzed by liver microsomes prepared directly from human livers (i.e., ex vivo rates). Although a few ex vivo samples exceed 12 or Cytochrome P450 induction in cultured human even 15 nmol/mg protein/min, it would appear that human hepatocytes hepatocytes in culture can, for all practical purposes, synthesize as much CYP3A4 in vitro as can be synthesized We recently reported on the induction of multiple CYP in vivo. In vehicle-treated or untreated hepatocytes, the so- enzymes by prototypical inducers in 62 preparations of called control CYP3A4 activity can vary enormously, cultured human hepatocytes (Madan et al., 2003). After a which contributes substantially to the variation in fold 2-day adaptation period, primary cultures of human hep- induction. Given that the upper limit of testosterone 6h- atocytes were treated with vehicle [dimethylsulfoxide hydroxylase activity in microsomes prepared from culture (DMSO), 0.1%, v/v] or one of three prototypical inducers, hepatocytes is about 12 (possibly 15) nmol/mg protein/ namely, h-naphthoflavone (33 AM), phenobarbital (250 min, treatment of hepatocytes with a control activity of 5 AM), and rifampin (20 AM). h-Naphthoflavone activates nmol/mg protein/min would result in no more than a 2- to the arylhydrocarbon (Ah) receptor and thereby induces 3-fold induction of CYP3A4 activity, whereas hepatocytes CYP1A2. Phenobarbital primarily activates the constitu- with a control activity of 0.5 nmol/mg protein/min might tive androstane receptor (CAR), and rifampin primarily be inducible more than 20-fold. The factors contributing to activates the pregnane X receptor (PXR). These two such diverse control rates have not been identified. Nor is receptors control the expression of CYP2B6, CYP2C9, it known why the ‘‘induced rates’’ do not always reach the A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 205

Fig. 12. The effects of age, gender, and ethnicity on CYP1A2 induction in human hepatocytes. same ceiling level (e.g., approximately 12 nmol/mg pro- results also demonstrate that 20 AM rifampin is generally tein/min for testosterone 6h-hydroxylation for microsomes more effective than 250 AM phenobarbital at inducing from rifampin-induced hepatocytes). CYP3A4. This is because 20 AM rifampin represents a maximal inducing concentration (i.e., higher concentrations Effects of age, gender, and ethnicity on CYP1A2 induction of rifampin cause no further increase in CYP3A4 activity), in human hepatocytes whereas 250 AM phenobarbital is submaximal, as previ- ously reported (LeCluyse et al., 2000). The effects of age, gender, and ethnicity on the induction of CYP1A2 in human hepatocytes treated with h-naphtho- Effects of gender on CYP2B6, CYP2C9, and CYP2C19 flavone are shown in Fig. 12. For many groups, the sample induction in human hepatocytes size is small, so care must be exercised in interpreting these data. With this limitation in mind, the results in Fig. 12 Fig. 14 summarizes the effects of gender on the suggest that induction of CYP1A2 by h-naphthoflavone is induction of CYP2B6, CYP2C9, and CYP2C19 in human not influenced by the age, gender, or ethnicity of the liver hepatocytes treated with phenobarbital or rifampin. The donor. small sample size preclude definitive conclusions from being drawn, but the results reveal that induction of Effects of age, gender, and ethnicity on CYP3A4 induction CYP2B6, CYP2C9, and CYP2C19 can be measured more in human hepatocytes or less to the same extent in hepatocytes from both male and female donors. When microsomes from cultured The effects of age, gender, and ethnicity on the induc- hepatocytes are compared with those prepared directly tion of CYP3A4 in human hepatocytes treated with phe- from human livers in terms of absolute rates (pmol/mg nobarbital or rifampin are shown in Fig. 13. Once again, protein/min), microsomes from control hepatocytes tend to for some groups, the sample size is small, so care must be have below average CYP2B6, CYP2C9, and CYP2C19 exercised in interpreting these data. With this caveat in activity. Microsomes from induced hepatocytes tend to mind, the results in Fig. 13 suggest that induction of have above average CYP2C9 activity (suggesting CYP3A4 by phenobarbital or rifampin is not influenced CYP2C9 is synthesized in vitro close to the upper limit by the age, gender, or ethnicity of the liver donor. The in vivo), but they tend to have average CYP2C19 activity

Fig. 13. The effects of age, gender, and ethnicity on CYP3A4 induction in human hepatocytes. 206 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209

Fig. 14. The effects of gender on CYP2B6, CYP2C9 and CYP2C19 induction in human hepatocytes; enzymatic rates are presented as pmol/mg protein/min. and below average CYP2B6 activity (compare Fig. 14 another important anatomical change in cirrhotic livers and with Table 1). It would appear, therefore, that induction is characterized by a loss of endothelial fenestrae, together of CYP2B6 and possibly CYP2C19 in vitro does not with the formation of a basal lamina beneath the endothelial reach the upper limit of synthesis observed in vivo, which cells, and the deposition of collagen in the space of Disse. In contrasts with the situation observed with CYP3A4 and rats with chemically induced cirrhosis, impaired diffusion of CYP2C9. Nevertheless, inasmuch as the control and albumin into the space of Disse was associated with a induced levels of CYP2B6 and CYP2C19 are similarly decreased uptake of protein-bound substrates into hepato- affected, induction of these enzymes is readily detectable cytes, which could contribute to the impairment of drug in human hepatocytes. metabolism. A study of the steady-state hepatic clearance and single-pass hepatic uptake of propranolol in perfused Effects of cirrhosis on CYP3A4 induction in cultured human cirrhotic and normal livers concluded that impaired uptake hepatocytes of protein-bound propranolol, as a result of capillarization and intrahepatic shunts, contributed significantly to its Liver function, including drug metabolism, is impaired in impaired elimination in cirrhosis. These anatomical and liver cirrhosis. The sick liver, healthy hepatocyte,orintact functional studies suggest that cirrhotic livers may in fact hepatocyte theory of liver cirrhosis states that impairment of contain relatively normal hepatocytes (Branch, 1982; drug metabolism in the disease is the result of the combi- Branch and Shand, 1976; Popper et al., 1952; Reichen, nation of functional intrahepatic shunts, which effectively 1989; Wu et al., 1991). reduce blood flow to the lobules and a reduced number of We have examined the ability of rifampin to induce normally functioning hepatocytes (Branch, 1982; Branch CYP3A4 in primary cultures of hepatocytes prepared from and Shand, 1976; Popper et al., 1952; Reichen, 1989; Wu et histologically normal and cirrhotic liver. As shown in Fig. al., 1991). 15, the degree to which CYP3A4 is inducible in hepatocytes In cirrhosis, masses of connective tissue dissect the isolated from cirrhotic liver is comparable to that achieved lobular parenchyma of the liver forming septa (borders, in hepatocytes isolated from normal liver, although there is walls) that constrain the hepatocytes. Although there are considerable sample-to-sample variation in the magnitude of several schools of thought, the prevailing opinion appears to induction in both cases. These results are consistent with the interpret the build-up of connective tissue as a response to recent clinical finding that treatment of cirrhotic patients epithelial cell injury. Septa in human cirrhosis are portacen- with phenobarbital induces the metabolism of aminopyrine tral, and epithelial cells in both portal and hepatic veins, as due to induction of cytochrome P450 (as opposed to well as in hepatic artery, contribute to the network of vessels improvements in blood flow or liver perfusion) (Herold et found in septa. The septa in human cirrhotic liver contain a al., 2003). Overall, the results in Fig. 15 suggest that vast network of vessels scantly connected to the sinusoids of hepatocytes isolated from cirrhotic liver may be suitable the lobular and nodular parenchyma. Within the vascular for in vitro studies of CYP enzyme induction, although network, there are many anastomoses (shunts) between these in vitro studies have yet to be extended to enzymes various branches of the portal vein, hepatic veins, and other than CYP3A4. hepatic artery, which are responsible for reduction in blood Cirrhotic livers potentially represent a large source of flow through sinusoids and reduced oxygen supply to hepatocytes for studies of human cytochrome function individual hepatocytes. Capillarization of the sinusoids is and regulation. However, two factors complicate the use A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209 207

Fig. 15. CYP3A4 induction in human hepatocytes: normal vs. cirrhotic liver.

of cirrhotic livers. First, cirrhotic livers are never used for variables that are known to have clinically relevant effects transplantation, hence, they are not perfused immediately on CYP enzyme expression, such as cigarette smoking, following removal with cold preservation medium, which which induces CYP1A2, and alcohol consumption, which is expensive. This means that warm ischemia time for induces CYP2E1, are poor predictors of CYP enzyme cirrhotic livers can be unacceptable (>20 min) unless activity in individual samples of human liver microsomes special arrangements are made. Second, cirrhotic livers (i.e., there is a wide and largely overlapping range of are often sliced (much like a loaf of bread) by patholo- CYP1A2 activity in smokers and nonsmokers, and there gists to look for tumors and other macroscopic lesions. is a wide and largely overlapping range of CYP2E1 This is an impediment to liver perfusion. If these activity in alcohol drinkers and nondrinkers). Some differ- obstacles can be overcome, cirrhotic livers can provide ences (generally not statistically significant) in mean CYP near-normal hepatocytes, but in yields below those activity were observed among groups, and some of these obtained with histologically normal liver. reflect clinical findings. For example, liver microsomal With the exception of Fig. 15, none of the data presented CYP1A2 activity tended to be lower in females than in this review were obtained with cirrhotic livers or anything males, whereas CYP3A4 activity in hepatocytes tended but histologically normal livers. to be higher in females than males, which is consistent with certain clinical findings. However, from the perspec- tive of conducting routine drug metabolism studies in Conclusions vitro, the magnitude of these differences is inconsequen- tial, especially when one considers the wide intersample Overall, the data summarized in this review consistent- variation in CYP activities within any particular group ly point to the same conclusion, namely, that CYP examined. enzyme activity in human liver microsomes and hepato- An interesting observation that stemmed from the anal- cytes varies considerably from one sample to the next, ysis presented here concerns ethnic differences. Compared and there is similarly widespread variation in the induc- with those from Caucasians and African Americans, liver ibility of CYP enzymes in cultured human hepatocytes. microsomes from Hispanics have about twice the activity of This variation is observed in human liver samples from CYP2A6, CYP2B6, and CYP2C8 activity and about half males and females, young, middle-aged and elderly the activity of CYP1A2. It remains to be established donors, and in livers from Caucasians, African Ameri- whether this apparent ethnic difference is a consequence cans, and Hispanics. Therefore, these variables (i.e., of the relatively low number of Hispanic donors, or whether, gender, age, and ethnicity) provide no meaningful basis in general, the expression of certain CYP enzymes in for selecting human-derived materials for routine studies Hispanics differs from that in Caucasians and African of drug metabolism and enzyme induction in vitro. Even Americans to a clinically relevant extent. 208 A. Parkinson et al. / Toxicology and Applied Pharmacology 199 (2004) 193–209

Induction of CYP enzymes in freshly cultured hepato- Harris, R.Z., Benet, L.Z., Schwartz, J.B., 1995. Gender effects in pharma- cytes does not appear to be influenced by the gender or age cokinetics and pharmacodynamics. Drugs 50, 222–239. Herold, C., Ganslmayer, M., Ocker, M., Zopf, S., Gailer, B., Hahn, of the donor. Furthermore, hepatocytes isolated from cir- E.G., Schu, D., 2003. Inducibility of microsomal liver function may rhotic liver can be used for in vitro studies of enzyme differentiate cirrhotic patients with maintained compared with se- induction (at least CYP3A4), which supports the ‘‘healthy verely compromised liver reserve. J. Gastroenterol. Hepatol. 18, hepatocyte, sick environment’’ hypothesis of liver cirrhosis. 445–449. In closing, it is worth saying a few words about a Hoch, U., Ortiz De Montellano, P.R., 2001. Covalently linked heme in cytochrome p4504a fatty acid hydroxylases. J. Biol. Chem. 276, potential conflict of interest. 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1996. Effects of freezing, thawing, and storing human liver microsomes Unimpaired induction of drug-metabolizing enzymes in hepatocytes on cytochrome P450. Arch. Biochem. Biophys. 331, 145–169. isolated from rats with micronodular cirrhosis. Can. J. Physiol. Phar- Popper, H., Elias, H., Petty, D.E., 1952. Vascular pattern of cirrhotic liver. macol. 69, 426–436. Am. J. Clin. Pathol. 22, 717–729. Wynne, H.A., Cope, L.H., Mutch, E., Rawlins, M.D., Woodhouse, K.W., Rasmussen, B.B., Brix, T.H., Kyvik, K.O., Brøsen, K., 2002. The interin- James, O.F.W., 1989. The effect of age upon liver volume and apparent dividual differences in the 3-demethylation of caffeine alias CYP1A2 is liver blood flow in healthy man. Hepatology 9, 297–301. determined by both genetic and environmental factors. Pharmacoge- netics 12, 473–478. A personal tribute to Ed Bresnick Reichen, J., 1989. Hepatic spaces and transport in the liver. In: Petzinger, I first met Ed Bresnick when I was a graduate student at the University of E., et al. (Eds.), Hepatic Transport of Organic Substances. Springer- Guelph in Ontario, Canada. Ed was our invited seminar speaker, and I shall Verlag, Berlin, pp. 45–56. never forget the impact of his talk. To appreciate why, I must point out that, Ryan, D.E., Levin, W., 1990. Purification and characterization of hepatic before going to Canada, I graduated from the University of Surrey in microsomal cytochrome P450. Pharmacol. Ther. 45, 153–239. England, and I did so in 1977. Where I got my degree is less important than Schmucker, D.L., Woodhouse, K.W., Rose, K., Wang, M.S., Wynne, H., when I got my degree, at least for the purpose of this story. Why? Because James, O.F., McManus, M., Kremers, P., 1990. Effects of age and in the mid-1970s, the molecular biology juggernaut we take for granted gender on in vitro properties of human liver microsomal monoxyge- today was only just picking up steam. Consequently, my undergraduate nases. Clin. Pharmacol. Ther. 48, 365–374. courses in biochemistry did not cover genetic cloning and all of the other Schuetz, E.G., Furuya, K.N., Schuetz, J.D., 1995. Interindividual variation molecular biology techniques that have revolutionized the life sciences. On and expression of P-glycoprotein in normal human liver and secondary the other hand, where I got my undergraduate training was important in hepatic neoplasms. J. Pharmacol. Ther. 275, 1011–1018. terms of my interest in attending Ed’s lecture on cytochrome P450 because Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., Guengerich, F.P., 1994. it was at the University of Surrey that I received my initial indoctrination in Interindividual variations in human cytochrome P-450 involved in the xenobiotic metabolism under the chairmanship of Dennis Parke, who, sad oxidation of drugs, carcinogens and toxic chemicals: studies with liver to say, also passed away recently. microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. I remember Ed’s talk because it sounded so visionary, which it was, although Ther. 270, 414–423. at the time it seemed also to be skirting on the edges of the twilight zone, if Sonderfan, A.J., Arlotto, M.P., Dutton, D.R., Parkinson, A., 1987. Regu- truth be told. He spoke of isolating the mRNA encoded by different lation of testosterone hydroxylation by rat liver microsomal cytochrome cytochrome P450 genes, of converting these mRNA species back into P-450. Arch. Biochem. Biophys. 255, 27–41. DNA, and of expressing these P450 ‘‘genes’’ in bacteria, where they could Tanaka, E., 1999. Gender-related differences in pharmacokinetics and their be produced ad infinitum. Today, such cloning is taken for granted. Back clinical significance. J. Clin. Pharm. Ther. 24, 339–346. then,talkofsuchthingswasspellbinding.Edwentontotalkabout Tucker, G.T., Houston, B., Huang, S.-M., 2001. Optimizing drug develop- combining parts of different P450 genes to make hybrid P450 enzymes; ment: strategies to assess drug metabolism/transporter interaction po- enzymes that have never existed before, with who-knew-what properties. tential—towards a consensus. Clin. Pharmacol. Ther. 70, 103–114. Perhaps they could be used to clean up oil spills, or make new chemicals, Vistisen, K., Loft, S., Poulsen, H.E., 1991. Cytochrome P450 1A2 activity even drugs. To hear Ed tell it, the sky was the limit. Much of what Ed talked in man measured by caffeine metabolism: effect of smoking, broccoli about has come true, through his own work and that of many others. But that and exercise. Adv. Exp. Med. Biol. 283, 407–411. is not what I relish about Ed’s talk. It was his enthusiasm, which was Wacher, V.J., Wu, C.Y., Benet, L.Z., 1995. Overlapping substrate specific- infectious and inspirational. There are not many who can extend formative ities and tissue distribution of cytochrome P450 3A and P-glycoprotein: ideas several steps into the future (at least not accurately), but Ed was one of implications for drug delivery and activity in cancer chemotherapy. them. Perhaps the following will put Ed’s talk in perspective: If you went to Mol. Carcinog., 129–134. graduate school over 30 years ago, how many talks by invited speakers can Waxman, D.J., Pampori, N.A., Ram, P.A., Agrawal, A.K., Shapiro, B.H., your remember? I can’t remember many, but I remember Ed’s talk vividly. 1991. Interpulse interval in circulating growth hormone patterns regu- Ed was collaborating with Wayne Levin, Dene Ryan, and Paul Thomas lates sexually dimorphic expression of hepatic cytochrome P450. Bio- (Allan Conney’s group) by the time I went to Hoffmann-La Roche for my chemistry 88, 6868–6872. postdoctoral training in the early 1980s, and it was then I got to meet Ed Wolbold, R., Klein, K., Burk, O., Nu¨ssler, A.K., Neuhaus, P., Eichelbaum, socially. He had a great sense of humor. With that in mind, I think he will M., Schwab, M., Zanger, U.M., 2003. Sex is a major determinant of forgive me for the following. Ed, you will be missed, although as long as CYP3A4 expression in human liver. Hepatology 38, 978–988. the image of Colonel Sanders adorns every Kentucky Fried Chicken outlet, Wu, Z.Q., Piche, D., Vallieres, S., Huet, P.M., Gascon-Barre, M., 1991. you will never be forgotten.