Platinum Anticancer Drugs Modulate P-450 Mrna Levels and Differentially Alter Hepatic Drug and Steroid Hormone Metabolism in Male and Female Rats1

[CANCER RESEARCH 52, 540-547. February 1, 1992] Platinum Anticancer Drugs Modulate P-450 mRNA Levels and Differentially Alter Hepatic Drug and Steroid Hormone Metabolism in Male and Female Rats1

Gerald A. LeBlanc, Scott S. Sundseth, Georg F. Weber, and David J. Waxman2

Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer Institute, Howard Medical School, Boston, Massachusetts 02115 [G. A. /.., S. S. S.. G. F. W., D. J. W.], and Department of Toxicology. North Carolina State University, Raleigh, North Carolina 27695 [G. A. L.J

ABSTRACT terone levels (3, 4). Cisplatin can also alter gonadal function (5), gonadotropin levels (6), and steroid hormone levels (6) in Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochromes P-450 and other microsomal patients undergoing cancer chemotherapy. Furthermore, cispla enzymes involved in steroid hormone and drug metabolism (G. A. Le tin chemotherapy can result in sterility, which presents a major Blanc, and D. J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). concern, since individuals treated with this drug for testicular The present study uses the rat model to evaluate the differential effects cancer are generally in their early reproductive years (7). of cisplatin treatment on liver microsomal enzymes between genders, and Many of the liver P-450-dependent hydroxylase enzymes also examines whether the modulation of enzyme activities by cisplatin whose expression is altered after cisplatin treatment participate and its analogues involves changes in P-450 gene expression. While in the metabolism of diverse lipophilic drugs, including cancer cisplatin treatment of male rats caused a severalfold increase in female- chemotherapeutic agents (8, 9). In this manner, cisplatin treat predominant hepatic enzymes, including testosterone 5a-reductase and ment could also have significant effects on the metabolism of testosterone 7a-hydroxylase (P-450 form 2A1), it partially decreased the other drugs that are administered with cisplatin in combination expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of chemotherapy. Such an effect was recently demonstrated in the rat model, whereby P-450 form 2C11, a major contributor to circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed hepatic microsomal activation of cyclophosphamide in male that the effects of cisplatin on P-450-catalyzed steroid hydroxylase rats, was suppressed after cisplatin treatment and the capacity activities in both male and female rats are primarily operative through for hepatic cyclophosphamide metabolism was consequently the drug's effects on P-450 mRNA expression. P-450-dependent cyclo- compromised (10). Such an effect could decrease the rate of phosphamide activation was significantly compromised in male rats after cyclophosphamide activation and perhaps alter its efficacy (9, cisplatin administration; however, this activity was not altered in cispla- 11). tin-treated females. This sex-dependent effect of cisplatin was due to its In the present study, we examined the effects of cisplatin on suppression of P-450 form 2C11, a male-specific P-450 that is a major steroid hormone and drug metabolizing enzymes and on indi contributor to microsomal cyclophosphamide bioactivation in male rat vidual liver P-450 mRNAs and compared these effects in adult liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did male and female rats. Also studied were the therapeutically not have significant effects on hepatic P-450 expression. Together, these active cisplatin analogues carboplatin and iproplatin, which findings demonstrate that the response of rat liver to cisplatin-induced have much reduced nephrotoxicity and neurotoxicity as com changes in hepatic P-450 enzyme profiles and cyclophosphamide bioac pared to cisplatin, and have myelosuppression as the dose- tivation capacity differs between the sexes, and in addition, these effects limiting toxicity (12-14). Results presented demonstrate that: can be minimized by use of carboplatin in place of cisplatin. (a) the effects of cisplatin on steroid hormone and drug metab olism in female rats are different from those observed in males; INTRODUCTION (Â¿>)theseeffects of cisplatin occur at a pretranslational level; and (c) these effects can be avoided by use of carboplatin in Cisplatin [eM-Pt(NH3)2Cl2] is widely used for the treatment place of cisplatin. of a variety of cancers, including gonadal and head and neck tumors (1). Cisplatin-based chemotherapy is complicated by a MATERIALS AND METHODS number of undesirable side effects, including nausea, renal toxicity, and neurotoxicity (1). Recent studies have demon Animal Treatments. Adult male and female Fischer 344 rats (Harlan- strated that male rats treated with cisplatin also undergo severe Sprague Dawley, Inc., Frederick, MD) were 9 weeks old at the beginning and prolonged changes in the expression of hepatic reducÃase of treatment and weighed 200 Â±15 g (SD) (males) or 144 Â±5 g and P-450-dependent hydroxylase enzymes that metabolize (females). Cisplatin (Johnson-Matthey), dissolved in 0.9% NaCl, was administered as a single i.v. injection at a dose of 0.72 mg/100 g body steroidal and drug substrates (2). The effects of cisplatin on these androgen-responsive enzymes are largely, though perhaps weight. This dose was previously shown to have significant effects on hepatic steroid metabolism in male rats (2). Cisplatin analogues (kindly not entirely, due to the associated suppression of circulating provided by Dr. Donald H. Picker and Dr. Michael J. Abrams, Johnson- testosterone (2). Cisplatin suppresses the activity of testicular Matthey, Inc., Westchester, PA) were administered as single i.v. injec steroid 17Â«-hydroxylase (P-450 17), a key enzyme in the andro- tions to male rats: carboplatin (JM-8) and iproplatin (JM-9) at 3 mg/ gen biosynthesis pathway, and this suppression has been impli 100 g body weight, and transplatin at 0.72 mg/100 g body weight. cated as causative for the observed reduction in serum testos- Dosages of these compounds were based upon their relative toxicities as compared to cisplatin (12, 13). Rats were killed by cervical disloca Received 7722/91; accepted 11/12/91. tion after asphyxiation under ('()_â€¢atdesignated times after drug The costs of publication of this article were defrayed in part by the payment treatment. Livers were quickly excised, perfused with ice-cold 1.15% of page charges. This article must therefore be hereby marked advertisement in KC1(w/v), sliced, quick-frozen in liquid nitrogen, then stored at -80Â°C accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'Supported in part by Grant CA-49248 from the NIH (1991-1994) and until individual pieces were used for preparation of microsomes or by Grant CN-14 from the American Cancer Society (1989-1991) (D. J. W.). G. RNA. Blood samples (-1.5 ml) collected by cardiac puncture at the A. L. was supported by a Postdoctoral Fellowship from the National Cancer time the rats were killed were allowed to clot overnight at 4Â°C.Serum Institute (Award l F32-CA08259). was collected by centrifugation the following day and stored at â€”¿20Â°C. 2To whom requests for reprints should be addressed, at: Dana-Farber Cancer Institute, Room JF-525, 44 Binney Street, Boston, MA 02115. Liver Microsome Preparation and Enzyme Assays. Microsomes were 540

Table 1 OUgonucleotide probes used for P450 mRNA analysis Hybridization conditions*P-450

ProbeA.form" ntsc323-346 sequence5'-TGT-ATT-GTA-GGT-AGC-CTGTTC-GCC-3' Female-predominant P-450s 2A1 (3) ON-6 S'-CAT-ATA-ATT-AAT-ATTTTT-TGC-TAT-TTT-GTG-S' 2C7 (f) ON-14 454545 0 670-699 ON-12B.2C12^(2d) 1020 698-727945-974 S'-AAT-AGC-AGC-AAA-ATG-TTT-TGA-ATGTGT-CTT-S'S'-ATC-CAC-GTG-TTT-CAG-CAG-CAG-CAG-GAG-TCCO' Male-specific P-450s 2Cll(2c) ON-5 5' ACT-GCC-TTT-GTG-AAG-ATC CCA- ATA- AAA-TTC-3 ' 3A2 (2a) ON-3 45 10 1528-1557 4A2ON-30C. 454045Formamide1010020cDNA 1691-1710586-607 5'GTG-TCT-GGA-GT-3'5'-AAA-TTT-TCA-TGT-TCT-CAT-TGA-G-3' GCT GGG-AAG Sex-independenl mRNAs 2C6(PB1) ON-4 a-Tubulin ON-50â€¢c45 39-66OligonucleotideS'-GAC-ATC-TTT-GGG-GAC-CAC-ATC-ACC-ACG-S' " P-450 form designations according to Nebert et al. (48), with trivial P-450 designations used in earlier studies from this laboratory (2,10, 21) shown in parentheses. The sex dependence of the expression of these rat P-450s is summarized (21) and is also apparent from Figs. 2 and 3. * Hybridization for Northern blot analysis was carried out as specified in "Materials and Methods," with the hybridization temperature (40* or 45'C) and formamide concentration (0-20%, v/v) adjusted for each Oligonucleotide probe, as indicated. These values are based on the length and (G - C) content of each probe, as described elsewhere (20). c The Oligonucleotide probes listed correspond to the complements of the corresponding P-450 cDNAs over the nucleotide intervals shown (nts). Nucleotide sequences are numbered from the initiator methionine codon, with exception of the a-tubulin probe ON-50. which is numbered according to the rat n-tubulin 3'-end cDNA clone pT25 (49). All of the P-450 probes are complementary to coding region sequences, with the exception of the 3A2 and 4A2 probes, which are complementary to 3'-untranslated sequences. d Female-specific expression.

on NENSORB 20 columns (DuPont-New England Nuclear) (18). Se quences of the individual probes and the conditions used for Northern ,5Â«-R blot hybridizations of each probe are shown in Table 1. RNA Isolation and Northern Blot Analysis. Total RNA was isolated from portions of individual rat livers using a guanidinium thiocyanate- u phenol-chloroform extraction method (19) with modifications (20). o' E 8-- RNA samples from individual livers were fractionated by electropho- resis under denaturing conditions in 1% agarose gels containing 40 mM 3-(/V-morpholino)propanesulfonic acid, 10 mM sodium acetate (pH 7.0), 0.66 Mformaldehyde, and 1 mM EDTA. The RNA was transferred 8. T, 7a-OH to nylon membranes (GeneScreen; DuPont-New England Nuclear) by 00 capillary transfer in lOx standard saline-citrate [lx standard saline- citrate = 15 mM sodium citrate (pH 7.0), 150 mM NaCI] and the RNA then fixed to the filters by UV cross-linking. Prehybridizations and hybridizations in buffers containing 0-20% formamide at 40 or 45Â°C, Days After Cisplatin as specified in Table 1 for individual Oligonucleotide probes, and high Fig. 1. Hepatic testosterone 5a-reductase (T,5a-R; â€¢¿)and7n-hydroxylase stringency washings were performed using methods described elsewhere activities (T,7a-OH, O) in female rats 1.5, 3, and 7 days after a single i.v. injection (20). The washed blots were air dried and exposed to Kodak XAR-5 of cisplatin at 0.72 mg/100 g body weight. Drug treatment, preparation of liver film with 1 or 2 intensifying screens at â€”¿80Â°Cfor1-5 days. Northern microsomes, and enzymatic analyses were carried out as described in "Materials and Methods." blots were stripped and then reprobed sequentially with additional Oligonucleotide probes (20). The rat a-tubulin Oligonucleotide probe ON-50 (Table 1) was used as a control to verify RNA load consistency prepared at 0-4Â°Cfrom individual rat livers by calcium precipitation, and integrity. The a-tubulin mRNA was found to be unaffected by as detailed elsewhere (2). Microsomal testosterone hydroxylase activi cisplatin treatment (data not shown). ties were determined using '""(labeled steroid substrate and a thin layer Estradici and Testosterone Measurements. Total serum estradici and chromatography assay (15). Microsomal testosterone 5a-reductase ac testosterone levels were measured by solid phase radioimmunoassay tivity was measured as described (2), except that testosterone was used using commercially available kits (Diagnostic Products Corporation, as substrate in place of androstenedione. Liver microsomal activation Los Angeles, CA). of cyclophosphamide was determined by a fluorometric assay after derivatization of acrolein with 3-aminophenol as described by Alarcon RESULTS (16) with modifications (17). Briefly, 2 HIM cyclophosphamide was incubated for 60 min at 37Â°Cin 0.2 ml containing 1 mM NADPH, 0.1 Effects of Cisplatin on Testosterone Metabolism in Female M KPi (pH 7.4), 0.1 mM EDTA, 5 mM semicarbazide HC1, 20% Rat Liver. Cisplatin treatment of adult male rats markedly glycerol, and 0.5 mg/ml microsomal protein. Protein was then precip suppresses several male-specific hepatic testosterone hydroxyl itated with zinc sulfate and barium hydroxide under acidic conditions, ase activities but elevates testosterone 7a-hydroxylase and tes and the supernatant was heated at 100Â°Cfor 20 min with 3-amino tosterone 5Â«-reductase activities from the low levels present in phenol and hydroxylamine (17). After cooling to room temperature in male rat liver to levels that are almost as high as those found the dark, the fluorescence of the samples was read (excitation at 350 nm and emission at 515 nm) using a Perkin-Elmer MPF-4 spectrofluo- in females (2). Since these latter 2 activities correspond to the rometer. All analytical data are expressed as mean Â±SD for 3-4 2 major pathways of testosterone metabolism in female rat liver individual rats per treatment group, unless indicated otherwise. (21), we examined the effects of cisplatin on these activities in Oligonucleotides. Gene-specific probes for individual P-450 mRNAs female rats. Liver microsomal testosterone 5Â«-reductase activ were synthesized, purified by high-performance liquid chromatography, ity was reduced by ~30% over a 7-day period after cisplatin 5'-labeled with [-x-32P]ATPby T4 polynucleotide kinase, then purified treatment, whereas testosterone 7a-hydroxylase activity was 541

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. IMPACT OF CISPLATIN ON HEPATIC GENE EXPRESSION decreased to a smaller extent that did not reach statistical Table 2 Serum estradiol and testosterone levels in cisplatin-treated female rats Rats were administered a single injection of cisplatin on day 0 and were significance (Fig. 1). Thus, microsomal enzyme activities to subsequently killed on the days indicated, as described in "Materials and Meth ward testosterone that are significantly elevated in male rats in ods". response to cisplatin treatment (Ref. 2 and data not shown) Day0 (pg/ml)24.0 (ng/ml)Â°<0.15 undergo small decreases in female rats given the same dose of Â±2.0* cisplatin. 1.5 8.6 Â±6.0 <0.15 Influence of Cisplatin on P-450 inKNA Levels. Rat liver 3 <5.0 <0.15 microsomal testosterone 7a-hydroxylation is catalyzed by P- 6Estradiol <5.0Testosterone <0.15 Â°Testosterone levels determined in parallel analyses of serum from untreated 450 form 2A1 (21). P-450 2A1 mRNA levels were therefore male rats were 3.9 Â±0.5 ng/ml. measured in livers from cisplatin-treated female and male rats * Mean Â±SD values for n = 3 rats/group. to determine whether the gender-dependent effects of cisplatin on testosterone 7a-hydroxylase activity are due to a differential modulation of P-450 2A1 mRNA levels. In accordance with response is qualitatively similar to that exhibited by P-450 2A1 the microsomal 7a-hydroxylase activity measurements (Fig. 1), mRNA (Fig. 2A). Cisplatin treatment also led to a partial P-450 2A1 mRNA levels in cisplatin-treated female rats were decrease in the expression of the female-specific P-450 2C12 somewhat reduced 7 days after drug administration (Fig. 2A, in the drug-treated female rats (Fig. 2C). In contrast to P-450s Lanes 14 and 75 versus Lanes 8-10), while in the males P-450 2A1 and 2C7, however, the expression of P-450 2C12 was not 2A1 mRNA levels were significantly elevated (Fig. 2, Lanes 5- induced in males treated with cisplatin (Fig. 2C). P-450 2C6 7 versus Lanes 1 and 2). Overall, cisplatin treatment resulted in mRNA, which is present at a similar level in male and female hepatic levels of P-450 2A1 mRNA (Fig. 2A) and P-450 2A1- rat liver, was not significantly affected by cisplatin treatment dependent testosterone 7<*-hydroxylase activity (data not (Fig. 2D). shown) that were very similar in the males and the females. Since P-450s 2A1, 2C7, and 2C12 each require estrogen for The observed modulation by cisplatin of liver P-450 2A1 full expression in female rats (21-24), we examined whether mRNA led us to investigate the effects of cisplatin on several circulating estradiol levels were altered in cisplatin-treated fe other female-predominant liver P-450 mRNAs. P-450 2C7, like male rats. Indeed, serum estradiol was found to be severely P-450 2A1, is expressed in female rat liver at a severalfold reduced after cisplatin treatment, whereas serum testosterone higher level than in males (e.g., Fig. 2B, Lanes 8-10 versus was undetectable in these female rats (Table 2). These findings Lanes 1 and 2). Cisplatin treatment decreased P-450 2C7 suggest that estrogen depletion may be an important cause of mRNA in female rats, whereas in males P-450 2C7 mRNA the observed decreases in these P-450s in cisplatin-treated levels were elevated after cisplatin treatment (Fig. 2B). This female rats.

MALE FEMALE

7 14 O DAYS A. 2A1 ; titfft B. 2C7 ... -

C. 2C12 â€¢¿â€¢-â€”-

D. 2C6 <*â€¢Â»-

1 2 4 5 6 7 8 9 10 11 12 13 14 15 Fig. 2. mRNA levels of the female-predominant P-450s 2A1 and 2C7 (A and B), the female-specific P-450 2C12 (C), and the gender-independent P-450 2C6 (D) in male (Lanes 1-7) and female (Lanes 8-15) rats after a single i.v. injection of cisplatin on day 0. Shown are autoradiographs of a Northern blot analyzing total liver RNA (20 fig/lane) isolated from individual rats in each treatment group and probed sequentially with oligonucleotides specific for the indicated P-450 mRNAs (Table 1) as described in "Materials and Methods." The reduced signal in Lane 6 for P-450 2C6 mRNA (D) and for a-tubulin mRNA (data not shown) indicates that the RNA analyzed in this lane was underloaded as compared to the other samples. lim,h to the right of each panel, migration of the 18S rRNA marker. 542

7 14 DAYS

Fig. 3. mRNA levels of the male-specific P-45Ã›S2C11, 3A2, and 4A2 in male and fe male rat liver after a single i.v. injection of A. 2C11 cisplatin on day 0. These results were obtained by reprobing the same Northern blot used for the analyses shown in Fig. 2. The P-450 2C6 signals shown in Fig. 2D are thus indicative of the RNA load and integrity for Fig. 3 as well B. 3A2 as Fig. 2.

C. 4A2

1 2 3 4 5 6 78 9 10 11 12 13 14 15

The suppressive effects of cisplatin on circulating estrogen suppression of male rat liver cyclophosphamide activation, levels and, accordingly, estrogen-dependent P-450 mRNAs in which was only partially reversed after 28 days (Fig. 4A), is female rats are analogous to the effects of cisplatin on circulat consistent with the observed loss of the cyclophosphamide 4- ing testosterone and testosterone-dependent P-450 protein lev hydroxylase P-450 2C11 in cisplatin-treated male rat liver (e.g., els in male rats (2). Experiments were therefore performed to Fig. 3). In females, however, P-450 2C11 is absent, and cyclo determine if cisplatin's suppressive effects on the expression of phosphamide activation is predominantly catalyzed by P-450 male-specific P-450s are also due to suppression of P-450 2C6 (10). Since P-450 2C6 is comparably expressed in both mRNA levels. P-450 2C11 mRNA became undetectable after males and females and is unaffected by cisplatin treatment (Fig. cisplatin administration to males (Fig. 3A), in accordance with 2D), cisplatin does not have an effect on microsomal cyclo the major loss of P-450 2C11 protein and activity in cisplatin- phosphamide activation in female rat liver (Fig. 4B). treated male rats (2). In contrast, P-450 3A2 and P-450 4A2 Effects of Cisplatin Analogues on Liver P-450-catalyzed Ste mRNAs, which are also expressed in rat liver in a male-specific roid and Drug Metabolism. The effects of cisplatin on liver P- fashion (Refs. 18, 25, and 26; also see Fig. 3, Lanes 1 and 2 450 activities were compared to those caused by 3 cisplatin versus Lanes 8-10), were induced in male rats 3 days after analogues: carboplatin and iproplatin, which are both active cisplatin administration but were subsequently suppressed to chemotherapeutic agents, and transplatin, an inactive isomer undetectable levels (Fig. 3, B and C). This transient induction of cisplatin. Iproplatin treatment of male rats resulted in a of these mRNAs is consistent with the observed increase in P- significant loss of liver microsomal testosterone 16a-hydroxyl- 450 3A2 protein levels and P-450 3A2-dependent testosterone ase activity (P-450 2C11-dependent; Ref. 21) and testosterone 6/3-hydroxylase activity 3 days after treatment of male rats with 60-hydroxylase activity (P-450 3A2-dependent) that was as cisplatin (2). None of the male-specific P-450 mRNAs was effective as that caused by cisplatin (Fig. 5, A and B). However, induced in female rats after cisplatin treatment (Fig. 3, Lanes iproplatin was less effective than cisplatin at inducing the 11-15). Together, these experiments establish that the multiple female-predominant enzymes testosterone 7Â«-hydroxylase (P- changes in hepatic P-450 activities and protein levels observed 450 2A1) and testosterone 5Â«-reductase (Fig. 5, C and D). in male rats after cisplatin treatment (2) are directly correlated Carboplatin and transplatin had no significant effect on these with changes in levels of the corresponding mRNAs. enzyme activities (Fig. 5). Since, in the case of iproplatin, Differential Effect of Cisplatin on Hepatic Microsomal Cyclo- suppression of the male-specific P-450 activities (Fig. 5, A and phosphamide Metabolism in Male and Female Rats. The differ B) is not tightly linked to induction of the female-predominant ential effect of cisplatin on liver P-450 expression in male and activities (Fig. 5, C and D), the inductive and suppressive effects female rats suggested that cisplatin might perturb P-450-cata- of these platinum-containing drugs on steroid metabolizing lyzed liver drug metabolism in a sex-dependent manner. This enzymes may involve independent mechanisms. was examined using the anti-cancer drug cyclophosphamide as Analysis of hepatic P-450 mRNA levels substantiated these a model substrate. Cyclophosphamide undergoes cytochrome differences in the effects of the platinum-containing drugs on P-450-catalyzed activation in the liver (11), and the specific liver P-450 expression (Fig. 6). P-450 2A1 mRNA levels were constitutive P-450 catalysts of this activation in the rat have elevated by cisplatin and iproplatin, but not by carboplatin or been identified as P-450s 2C11 and 2C6 (10). The absence of transplatin (Fig. 6A). P-450 2C12 mRNA, which is not nor P-450 2C11 in female rat liver accounts for the lower cyclo mally detectable in adult male rats, was also not induced by any phosphamide 4-hydroxylase (cyclophosphamide activation) ac of the platinum compounds (Fig. 6B). P-450 2C11 mRNA was tivity in liver microsomes isolated from female as compared to fully suppressed by cisplatin and iproplatin (Fig. 6C), an obser male rats (10). Cisplatin treatment of male rats reduced the vation consistent with the suppression by these drugs of P-450 rate of microsomal cyclophosphamide activation to female lev 2C11-mediated testosterone 16Â«-hydroxylaseactivity (Fig. 5/1). els (Fig. 4A), but had no effect on the rate of cyclophosphamide Carboplatin and transplatin had no effect on P-450 2C11 activation when administered to female rats (Fig. 4B). This mRNA, in accordance with the activity measurements shown 543

A. MALE Among the cisplatin analogues tested, iproplatin significantly suppressed cyclophosphamide 4-hydroxylase activity in liver 2.5 microsomes prepared from the drug-treated rats, whereas car boplatin and transplatin had only small effects on this enzyme 2.0 activity (Fig. 7). These observations are also consistent with the WD suppressive effects of iproplatin, but not carboplatin or tran splatin, on P-450 2C11 mRNA levels, and suggest that while cisplatin and iproplatin may both decrease liver capacity to activate cyclophosphamide, this potentially significant drug interaction can be minimized, and perhaps avoided, through "o l O the use of carboplatin. B C 0.5 DISCUSSION Cisplatin treatment of male rats results in a partial femini-

6 12 18 24 30 zation in the expression of hepatic drug and steroid-metaboliz ing enzymes (2). Demonstrated effects of cisplatin on proteins DAYS AFTER CISPLATIN and cytosolic ligands (27-29), mRNA (30), and DNA (31-33), suggest several possible mechanisms by which this compound could alter hepatic drug and steroid hormone metabolism. The present demonstration that the major effects of cisplatin on male rat liver P-450 expression occur at a pretranslational level, B. FEMALE and involve suppression of male-specific P-450 mRNAs, such as 2C11, 3A2, and 4A2, concomitant with an elevation of female-predominant P-450 mRNAs, such as 2A1 and 2C7, supports the conclusion that these effects of cisplatin are largely due to its perturbation of the hormonal regulation of these enzymes, and are not due to direct interactions of the drug with P-450 proteins. The present P-450 mRNA analyses also sup port our previous observation (2) that the male-specific P-450

A. 16a-OHose 4.0

2.0

1.5 3.0 4.5 6.0 7.5 0.0 B. 60-OHose 0.8 DAYS AFTER CISPLATIN en E Fig. 4. Hepatic microsomal cylcophosphamide activation catalyzed by liver C 0.4 microsomes isolated from male (A) and female (B) rats after a single injection of E cisplatin on day 0. Cyclophosphamide activation was analyzed using a fluorescent \ assay for acrolein formation as described in "Materials and Methods." The o experiment shown in B was terminated 7 days after cisplatin treatment. E 0.0 C. 7a-OHose in Fig. SA. However, P-450 3A2 mRNA and P-450 4A2 mRNA, although decreased by cisplatin 7 days after drug treatment (Fig. 6, Lanes 2 and 3; also see Fig. 3), were increased by iproplatin, as well as carboplatin and transplatin, at this same time point (Fig. 6, D and E, Lanes 4-8). Since cisplatin effected o. co a transient elevation of these 2 mRNAs that was detectable at 3 days (Fig. 3), the apparent elevation of these mRNAs by iproplatin at the 7-day time point (Fig. 6, Lanes 6 and 7 versus Lane 1) may reflect an inductive effect that is similar to, albeit delayed, or perhaps longer-lasting than the one caused by cisplatin. It should be noted, however, that the induction of P- CIS CBO IPR TR 450 3A2 mRNA by iproplatin contrasts with the observed suppression of P-450 3A2-dependent testosterone 6/3-hydrox- DRUG TREATMENT Fig. 5. Hepatic microsomal testosterone hydroxylase and reducÃaseactivities ylase activity in the same iproplatin-treated rats (Fig. 6D versus in sham-injected male rats (â€”)or male rats 7 days after treatment with cisplatin Fig. 5B). This suggests that iproplatin could inhibit P-450 3A2 (CIS), carboplatin (CBO), iproplatin (IPR), or transplatin (TR). Hydroxylation enzyme activity through mechanisms that involve translational of testosterone at the 16a, 6/3, and 7a positions (A-C, respectively), is catalyzed by P-450 forms 2C11, 3A2, and 2A1, respectively, in uninduced adult male rat effects or perhaps direct interactions with the P-450 3A2 liver (21). The activities shown in A and B are male-specific; those in C and /) are protein. female-predominant. 544

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. IMPACT OF CISPLATIN ON HEPATIC GENE EXPRESSION UT CIS CBO IPR TR testosterone is a positive regulator of pituitary growth hormone secretion (38, 39). Whereas cisplatin increased several female-predominant mRNAs and enzyme activities, including hepatic testosterone - 7a-hydroxylase and testosterone 5a-reductase activities when given to male rats, it did not increase, but rather decreased, A. 2A1 these activities in females. Similarly, suppression by cisplatin of hepatic microsomal cyclophosphamide bioactivation oc curred in male but not female rats treated with cisplatin. Thus, there is a sex difference in the responsiveness of rats to cisplatin- induced perturbations in drug- and steroid-metabolizing en B. 2C12 zymes. For several of these enzymes and mRNAs, female- predominant expression in part results from a positive regula tory role of the estrogen-stimulated continuous plasma growth hormone profile, which maintains their expression in female rat liver, and from the suppressive effect of the episodic growth hormone secretion profile of males (21-23), which is androgen- C. dependent (38, 39). This suggests that the decrease in these i n t activities in cisplatin-treated females may result from the loss of circulating estradiol (Table 2), whereas the increase in these activities in cisplatin-treated males is likely to be due to the decrease in serum testosterone (2, 3) and the resultant loss of D. 3,42 circulating growth hormone. Cisplatin-induced feminization of liver enzyme expression was not complete, insofar as the female-specific P-450 2C12 was not induced in cisplatin-treated male rats. As with the â€¢¿â€¢¿ female-predominant P-450s, expression of P-450 2C12 is pos E. 4A2 itively regulated by the effects of estrogen (23) and its associated stimulation of continuous growth hormone secretion (40). In contrast to the female-predominant enzymes, however, P-450 2C12 is not under negative control of adult androgen (41) and, accordingly, its expression is not induced in cisplatin-treated males. P-450 2C12 mRNA levels were, however, somewhat F. 2C6 decreased in cisplatin-treated female rats, an effect that may be due to the reduction in serum estradiol levels that follows drug treatment. Thus, in both males and females, cisplatin appears to decrease the expression of the sex-specific P-450 forms by 12345678 lowering the circulating levels of androgens and estrogens re Fig. 6. Northern blot analysis of hepatic P-450 mRNA levels 7 days after quired for full enzyme expression. That these effects of cisplatin treatment of male rats with the indicated platinum compounds (see Fig. 5 for drug designations). UT, sham-injected ("untreated") rat. Northern blot analysis are likely to be a consequence of the loss of circulating gonadal was as described in Fig. 2 using total RNA prepared from the same liver samples analyzed for enzyme activities in Fig. 5. Differences in 3A2 mRNA levels in Lane 6 versus Lane 7 reflect individual differences in the responsiveness of this mRNA to iproplatin (cf., consistency of responsiveness of 2A1, 2C11, and 2C6 mRNA to iproplatin). 2.0

3A2 is initially increased after cisplatin treatment but subse u quently is suppressed. This response contrasts that of a second Ã¶fl male-specific P-450, form 2C11, whose expression is progres e 1.5 D, sively decreased after cisplatin treatment (Ref. 2; Fig. 3). Studies O of the hormonal requirements for the expression of these 2 P- 450s have shown that whereas P-450 2C11 requires episodic < 1.0 S growth hormone secretion for full expression (34, 35), P-450 o o Vi e 3A2 is primarily under the suppressive influence of growth o C K O 5 hormone (36, 37). Accordingly, depletion of circulating growth U hormone by hypophysectomy leads to an increase in the expres S sion of P-450 3A2 (18, 36, 37), as well as P-450 4A2, which is K subject to similar regulation by growth howmone in male rat 0.0 liver (26). The initial increase in P-450s 3A2 and 4A2, but not 2C11, observed at the 3-day time point suggests the possibility UT CIS CARSO IPRO TRANS that circulating growth hormone levels may be severely depleted Fig. 7. Hepatic microsomal cyclophosphamide hydroxylase activity in male rats 7 days after treatment with cisplatin (CIS), carboplatin (CARBO), iproplatin during the initial 3 days after cisplatin administration. This (IPRO), or transplatin (TRANS), as described in "Materials and Methods." hypothesis is consistent with the observed loss of circulating Points, mean of duplicate activity determinations; bars, mean for the activities of testosterone in cisplatin-treated male rats (2, 3), insofar as two individual liver microsome preparations in each treatment group. 545

expression by cisplatin. Evidence for perturbations in the hormonal regula steroids is further supported by the observation that cisplatin tion of steroid-metabolizing enzymes. J. Biol. Chem., 263: 15732-15739, alters the profile of steroid reducÃaseand hydroxylase enzymes 1988. in a manner that is very similar to that elicited by adult Maines, M. D., and Mayer, R. D. Inhibition of testicular cytochrome P-450- dependent steroid biosynthesis by m-pkitmuni. J. Biol. Chem., 260: 6063- gonadeclomy (22, 23, 41). 6068, 1985. The high degree of sexual dimorphism in ihe expression of Maines, M. D., Sluss, P. M., and Iscan, M. cu-Platinum-mediated decrease drug and steroid-hormone metabolizing enzymes in rat liver in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis. Endocrinology, 126: 2398- and the differential response of these enzymes to the effects of 2406, 1990. cisplatin suggest that hepatic drug metabolism might be differ Wallace, W. H., Shalet, S. M., Crowne, E. C, Morris-Jones, P. S., Gatta- entially altered between cisplatin-treated males and females. maneni, H. R., and Price, D. A. Gonadal dysfunction due to cu-platinum. Med. Pediatr. Oncol., 17: 409-413, 1989. Indeed, although microsomal activation of cyclophosphamide LeBlanc, G. A., Kantoff, P. W., Ng, S. F., Frei, E IH, and Waxman, D. J. was significanlly suppressed after exposure of male rats to Hormonal perturbations in testicular cancer patients treated with cisplatin. cisplatin (Fig. 4A; Ref. 10), Ibis activation was not affected in Cancer (Phila.), in press, 1992. Drasga, R. E., Einhorn, L. H., Williams, S. D., Patel, D. N., and Stevens, E. similarly treated female rats. The primary catalysts of cyclo E. Fertility after chemotherapy for testicular cancer. J. Clin. Oncol., /: 179- phosphamide bioactivation in male rat liver are P-450 2C11 183, 1983. Wislocki, P. G., Miwa, G. T., and Lu, A. Y. H. Reactions catalyzed by the and P-450 2C6 (10). In females, where P-450 2C11 is absent, cytochrome P-450 system. In: W. B. Jakoby (ed.), Enzymatic Basis of cyclophosphamide aclivation is due primarily to P-450 2C6 Detoxification, Vol. 1, pp. 135-182. New York: Academic Press, 1980. (10). Since cisplatin ad minisi ration suppresses P-450 2C11, bul LeBlanc, G. A., and Waxman, D. J. Interaction of anticancer drugs with hepatic monooxygenase enzymes. Drug Metab. Rev., 20: 395-439, 1989. noi P-450 2C6, cisplatin treatmenl decreases hepalic cyclo 10. Clarke, I... and Waxman, D. J. Oxidative metabolism of cyclophosphamide. phosphamide bioaclivalion only in Ihe males. Identification of the hepatic monooxygenase catalysts of drug activation. Cancer Res., 49: 2344-2350, 1989. Sexual dimorphisms also exisl in drug and steroid hormone 11. Sladek, N. E. Therapeutic efficacy of cyclophosphamide as a function of its metabolism in humans, although the differences are much metabolism. Cancer Res., 32: 535-542, 1972. smaller than Ihose observed in rals (42-44). Il is Ihus conceiv 12. Reed, E., and Kohn, K. W. Platinum analogues. In: B. A. Chabner and J. M. Collins (eds.), Cancer Chemotherapy: Principles & Practice, pp. 465-490. able lhal effects of cisplatin on drug and sleroid hormone Philadelphia: J. B. Lippincott Co., 1990. melabolizing enzymes similar to Ihose observed in Ihe ral mighl 13. Bunn, P. A., Jr., Canetta, R., Ozols, R. F., and Rozencweig, M. (eds.), also occur in humans. Increased plasma dihydrotestoslerone, a Carboplatin (JM-8). Current Perspectives and Future Directions. Philadel phia: W. B. Saunders Company, 1990. leslosterone metabolile formed by sleroid 5a-reduclase, has 14. Pendyala, L., Cowens, J. W., Chheda, G. B., Dutta, S. P., and Creaven, P. been observed in humans undergoing cisplalin-based ireatment J. Identification of m-dichloro-bis-isopropylamine platinum (II) as a major metabolite of iproplatin in humans. Cancer Res., 48: 3533-3536, 1988. for testicular cancer (6). This finding is consislenl wilh ihe IS. Waxman, D. J. P450-catalyzed steroid hydroxylation. Assay methods and elevated steroid 5a-reduclase aclivily observed in male rals product identification by TLC. Methods Enzymol., 206: 462-476, 1991. treated with cisplatin (Ref. 2; Fig. 5). The possibility thai 16. Alarcon, R. A. Fluorometric determination of acrolein and related com pounds with m-aminophenol. Anal. Chem., 40:1704-1708, 1968. hepalic sleroid and drug metabolism may be altered in patients 17. Masurel, D., Houghton, P. J., Young, C. L., and Wainer, I. W. Efficacy, undergoing treatment with cisplatin would be of grealesl con toxicity, pharmacokinetics, and in vitro metabolism of the enantiomers of cern in Ihe case of high dose Irealmenl regimens (45), in which ifosfamide in mice. Cancer Res., 50: 252-255, 1990. 18. Waxman, D. J., Ram, P. A., Nolani, G., LeBlanc, G. A., Alberta, J. A., patients receive cisplatin al doses comparable lo Ihose lhal Morrissey, J. J., and Sundseth, S. S. Pituitary regulation of the male-specific markedly aller hepalic drug and sleroid metabolism in ihe ral steroid 6 ^-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretrans- model. lational level. Mol. Endocrinol., 4:447-454, 1990. Comparison of cisplalin lo Ihe inaclive analogue Iransplalin 19. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by and Ihe iherapeulically aclive analogues carboplalin and ipro acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 762: 156-159, 1987. platin revealed lhal only iproplatin alters Ihe pallern of hepalic 20. Waxman, D. J. Analysis of rat hepatic P450IIA and P450IIC subfamily P-450 mRNA and aclivily expression in a manner similar lo expression using catalytic, immunochemical. and molecular probes. Methods cisplalin. Circulaling lesloslerone levels are known lo be de Enzymol., 206: 249-267, 1991. 21. Waxman, D. J. Interactions of hepatic cytochromes P-450 with steroid creased in aduli male rals afler treatment with iproplatin, but hormones. Regioselectivity and stereospecificity of steroid hydroxylation and not carboplatin (46), a finding thai strenglhens our conclusion hormonal regulation of rat P-450 enzyme expression. Biochem. Pharmacol., 3 7:11-84, 1988. lhal lesloslerone depletion is cenlral lo ihe effects of cisplatin 22. Waxman, D. J., Morrissey, J. J., and LeBlanc, G. A.. Female-predominant and iproplatin on hepatic enzyme expression. The present rat hepatic cytochrome P-450 forms j (IIE1) and 3 (IIA1) are under hormonal findings also indicate thai ihe perturbation of drug-metaboliz regulatory controls distinct from those of the sex-specific P-450 forms. Endocrinology, 124: 2954-2966, 1989. ing P-450s can be minimized by use of carboplatin, which has Dannan, G. A., Guengerich, F. P., and Waxman, D. J. Hormonal regulation been inlroduced inlo the clinic as an active agent with much- of rat liver microsomal enzymes: role of gonadal steroids in programming, reduced nephrotoxicily compared lo cisplalin (12, 13). Al- maintenance and suppression of A4-steroid 5a-reductase, flavin-containing monooxygenase and sex-specific cytochromes P-450. J. Biol. Chem., 26/: Ihough cisplalin and carboplatin form idenlical DNA adducls, 10728-10735, 1986. ihe inaclivity of carboplatin with respecl lo effecls on liver 24. Sasamura, H., Nagata, K., Yamazoe, Y., Shimada, M., SanitÃ ,T., and Kalo, R. Effect of growth hormone on rat hepatic cytochrome P-450f mRNA: a enzyme expression suggesls lhal ihis drug is less able lo access new mode of regulation. Mol. Cell Endocrinol., 68: 53-60, 1990. Ihe testicular and/or cenlral sile(s) at which cisplatin binds lo 25. Gonzalez, F. J., Song, B-J., and Hardwick, J. P. Pregnenolone-16o-carbo- elicil ils effecls on hepalic P-450 expression (2,4). Carboplatin nitrile-inducible P-450 gene family: gene conversion and differential regula tion. Mol. Cell Biol., 6: 2969-2976, 1986. exhibils different pharmacokinetics, tissue distribution, and 26. Sundseth, S. S., and Waxman, D. J. Sex-dependent expression and clofibrate protein binding patterns than cisplalin, and Ihese factors appear inducibility of cytochrome P450 4A fatty acid u-hydroxylases. Male-specific likely lo conlribule lo ils reduced cenlral, renal, and gonadal ity of liver and kidney CYP4A2 mRNA and tissue-specific regulation by growth hormone and testosterone. J. Biol. Chem., in press, 1992. loxicilies (13, 27, 46, 47) as compared lo cisplalin. 27. Litterst, C. L. Cisplatinum: a review, with special reference to cellular and molecular interactions. Agents Actions, IS: 520-524, 1984. 28. Sharma, R. P., and Edwards, I. R. cis-Platinum: subcellular distribution and REFERENCES binding to cytosolic ligands. Biochem. Pharmacol., 32: 2665-2669, 1983. 29. Zelazowski, A. J., Garvey, J. S., and Hoeschele, J. D. In vivo and in vitro 1. Loehrer, P. J., and Einhorn, L. H. Cisplatin: diagnosis and treatment. Drugs binding of platinum to metallothionein. Arch. Biochem. Biophys., 229: 246- five years later. Ann. Intern. Med., 700: 704-713, 1984. 252, 1984. 2. LeBlanc, G. A., and Waxman, D. J. Feminization of rat hepatic P-450 30. Rosenberg, J., and Sato, P. Messenger RNA loses the ability to direct in vitro 546

peptide synthesis following incubation with cisplatin. Mol. Pharmacol., 33: regulation of cytochrome P-450 15d-apoprotein levels in rat liver. Endocri 611-616, 1988. nology, 117: 2085-2092, 1985. 31. Roberts, J. J., and Thomson, A. J. The mechanism of action of antitumor 41. Waxman, D. J., Dannan, G. A., and Guengerich, F. P. Regulation of rat platinum compounds. Prog. Nucleic Acids Res. Mol. Biol., 22:71-133,1979. hepatic cytochrome P-450: age-dependent expression, hormonal imprinting 32. Sherman, S. E., Gibson, D., Wang, A. H. J., and Lippard, S. J. X-ray and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry, 24: structure of the major adduci of the anticancer drug cisplatin with DNA: cis- 4409-4417, 1985. 42. Goble, F. C. Sex as a factor in metabolism, toxicily and efficacy of pharma- [Pt(NH3)2|d(pGpG)|]. Science (Washington DC), 230: 412-417, 1985. codynamic and chemotherapeutic agents. Adv. Pharmacol. Chemother., 13: 33. Reed, E., Litterst, C. L., Thill, C. C., Yuspa, S. H., and Poirier, M. C. cis- 173-252, 1975. Diamminedichloroplatinum(II)-DNA adduci formation in renal, gonadal, 43. Bennett, B. M., Twiddy, D. A. S., Moffat, J. A., Armstrong, P. W., and and tumor tissues of male and female rats. Cancer Res., 47: 718-722, 1987. Marks, G. S. Sex-related difference in the metabolism of isosorbide dinitrate 34. Zaphiropoulos, P. G., Mode, A., Norstedt, G., and Gustafsson, J-A. Regu following incubation in human blood. Biochem. Pharmacol., 32:3729-3734, lation of sexual differentiation in drug and steroid metabolism. Trends 1983. Pharmacol. Sci., 10: 149-153, 1990. 44. Pfaffenberger, C. D., and Horning, E. C. Sex differences in human urinary 35. Waxman, D. J., Pampori, N. A., Ram, P. A., Agrawal, A. K., and Shapiro, steroid metabolic profiles determined by gas chromatography. Anal. B. H. Interpuise interval in circulating growth hormone patterns regulates Biochem., SO: 329-343, 1977. sexually dimorphic expression of hepatic cytochrome P450. Proc. Nati. Acad. 45. Amman, K., Eder, J. P., Elias, A., Shea, T., Peters, W. P., Andersen, J., Sci. USA, 88: 6868-6872, 1991. Schryber, S., Henner, W. D., Finberg, R., Wilmore, D., Kaplan, W., Lew, M., et al. High dose combination alkylating agent preparative regimen with 36. Waxman, D. J., LeBlanc, G. A., Morrissey, J. J., Staunton, J., and Lapenson, autologous bone marrow support: The Dana-Farber Cancer Institute/Beth D. P. Adult male-specific and neonatally programmed rat hepatic P-450 Israel Hospital experience. Cancer Treat. Rep., 71: 119-125, 1987. forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone 46. Azouri. H.. Bidart. J. M., and Bohuon, C. In vivo toxicity of cisplatin and for expression. J. Biol. Chem., 263: 11396-11406, 1988. carboplatin on the Leydig cell function and effect of the human choriogona- 37. Yamazoe, Y., Murayama, N., Shimada. M., Yamauchi, K., Nagata, K., dotropin. Biochem. Pharmacol., 38: 567-571, 1989. Imaoka, S., Funae, Y., and Kato, R. A sex-specific form of cytochrome P- 47. Siddik, Z. H., Newell, D. R., Boxall, F. E., and Harrap, K. R. The compar 450 catalyzing propoxycoumarin O-depropylation and its identity with tes ative pharmacokinetics of carboplatin and cisplatin in mice and rats. tosterone 6(3-hydroxylase in untreated rat livers: reconstitution of the activity Biochem. Pharmacol.. 36: 1925-1932. 1987. with microsomal lipids. J. Biochem. (Tokyo), 104: 785-790, 1988. 48. Nebert, D. W.. Nelson, D. R., Coon, M. J., Estabrook. R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus. I. C., 38. Jansson, J. O., and Frohman, L. A. Differential effects of neonatal and adult Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., and Waxman, D. androgen exposure on the growth hormone secretory pattern in male rats. J. The P450 superfamily: update on new sequences, gene mapping, and Endocrinology, 120: 1551-1557, 1987. recommended nomenclature. DNA Cell Biol., 10: 1-14, 1991. 39. Jansson, J-O., Eden, S., and Isaksson. O. Sexual dimorphism in the control 49. Ginzburg, I., Behar, L., Givol, D., and Littauer, U. Z. The nucleotide of growth hormone secretion. Endocrine Rev., 6: 128-150, 1985. sequence of rat r///>/iutnhuliii: 3'-end characteristics, and evolutionary con 40. MacGeoch, C., Morgan, E. T., and Gustafsson, J. A. Hypothalamo-pituitary servation. Nucleic Acids Res., 9: 2691-2697, 1981.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. Platinum Anticancer Drugs Modulate P-450 mRNA Levels and Differentially Alter Hepatic Drug and Steroid Hormone Metabolism in Male and Female Rats

Gerald A. LeBlanc, Scott S. Sundseth, Georg F. Weber, et al.

Cancer Res 1992;52:540-547.

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