Clinical Cancer Research Targets Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reid, J. M.
Right arrow Articles by Ames, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reid, J. M.
Right arrow Articles by Ames, M. M.
Clinical Cancer Research Vol. 5, 2192-2197, August 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Metabolic Activation of Dacarbazine by Human Cytochromes P450: The Role of CYP1A1, CYP1A2, and CYP2E11

Joel M. Reid2, Mary J. Kuffel, Jennifer K. Miller, Robert Rios3 and Matthew M. Ames

Department of Oncology, Division of Developmental Oncology Research, Mayo Clinic, Rochester, Minnesota 55905


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dacarbazine (DTIC), a widely used anticancer agent, is inactive until metabolized in the liver by cytochromes P450 to form the reactive N-demethylated species 5-[3-hydroxy-methyl-3-methyl-triazen-l-yl]-imidazole-4-carboxamide (HMMTIC) and 5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide (MTIC). The modest activity of DTIC in the treatment of cancer patients has been attributed in part to lower activity of cytochromes P450 (P450) in humans when compared with rodents. Importantly, the particular P450 isoforms involved in the activation pathway have not been reported. We now report that the DTIC N-demethylation involved in MTIC formation by human liver microsomes is catalyzed by CYP1A1, CYP1A2, and CYP2E1. The most potent inhibitors of DTIC N-demethylation were {alpha}-naphthoflavone (CYP1A1 and CYP1A2), quercetin (CYP1A2), chlorzoxazone (CYP1A2 and CYP2E1), and di-sulfiram (CYP2E1). Antihuman CYP1A2 antiserum also inhibited DTIC N-demethylation. DTIC N-demethylation in a panel of 10 human liver microsome preparations was correlated with the catalytic activities for CYP1A2 (ethoxyresorufin O-deethylation and caffeine N3-demethylation) in the absence of {alpha}-naphthoflavone and with the catalytic activities for CYP2E1 (chlorzoxazone 6-hydroxylations) in the presence of {alpha}-naphthoflavone. DTIC metabolism was catalyzed by recombinant human CYP1A1, CYP1A2, and CYP2E1. The Km (Vmax) values for metabolism of DTIC by recombinant human CYP1A1 and CYP1A2 were 595 µM (0.684 nmol/min/mg protein) and 659 µM (1.74 nmol/min/mg protein), respectively. The CYP2E1 Km value exceeded 2.8 mM. Thus, we conclude that (a) CYP1A2 is the predominant P450 that catalyzes DTIC hepatic metabolism; (b) CYP2E1 contributes to hepatic DTIC metabolism at higher substrate concentrations; and (c) CYP1A1 catalyzes extrahepatic metabolism of DTIC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DTIC4 is a DNA-methylating agent with a broad spectrum of antitumor activity in mouse tumor models. Clinical activity of this agent in human malignancies is restricted to melanoma, Hodgkin’s disease, and sarcoma. DTIC is used in a curative regimen with Adriamycin, bleomycin, and vincristine (ABVD) for Hodgkin’s disease and is the most active single agent used to treat advanced melanoma and soft tissue sarcoma (1) .

The DNA methylation produced by DTIC is dependent upon oxidative N-demethylation by P450s (2 , 3) . Early investigators identified formaldehyde and AIC as products of DTIC N-demethylation by mouse and rat liver microsomal preparations (2 , 3) . The proposed cascade of DTIC metabolism and formation of the reactive methylating molecule is illustrated in Fig. 1Citation . The initial product of P450-catalyzed oxidation of DTIC, the carbinolamine HMMTIC, produces MTIC after elimination of formaldehyde. Rapid decomposition of MTIC yields the major plasma and urine metabolite AIC and the reactive species methane diazohydroxide, which produces molecular nitrogen and a methyl cation believed to be the methylating species. The importance of this pathway was confirmed in vivo by the recovery of 14CO2 in expired breath and HMMTIC, AIC, and [14C]methylguanine in urine after an i.p. dose of [14C-methyl]DTIC (4) . In human pharmacokinetic studies, metabolism was the predominant pathway for DTIC clearance. Plasma exposure to AIC was equivalent to that of DTIC, whereas urinary recovery of DTIC and AIC was low and highly variable.



View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Pathway of DTIC metabolism.

 
The P450s comprise a superfamily of enzymes that catalyze the metabolism of many drugs including several anticancer agents (5) A single isozyme is often predominant in the metabolism of a given drug (6) . The expression of P450 is often highly variable among individuals because of genetic, physiological, and environmental factors (6) . Metabolism by P450s may be inhibited or induced by concomitant drug treatment. The consequences of individual differences in P450 isozyme expression and drug interactions caused by coadministered drugs on DTIC antitumor activity and plasma clearance are presently unclear, and the limited published pharmacokinetic data (7 , 8) is insufficient to evaluate population variability of DTIC metabolism.

Rodent P450 isoforms may have different activity and substrate specificity when compared with their human homologues (9) , thereby contributing to species-specific differences in drug metabolism. The limited antitumor activity of DTIC in humans compared with mice has been attributed to reduced metabolism by human P450s as compared with rodent liver P450s (10) . This hypothesis, however, has been difficult to confirm because of poor stability of the active metabolite, MTIC.

Efforts to improve DTIC activity have focused on the development of analogues that improve the delivery of the methylating metabolites. For example, the recent clinical development of temozolomide—an analogue with demonstrated activity against metastatic melanoma (11) and high-grade astrocytoma (12) —was based on the presumption that greater and more reproducible exposure to MTIC could be achieved by the chemical hydrolysis of temozolomide at physiological pH rather than by the hepatic metabolism of DTIC (13) . These efforts do not, however, provide any guidance in how to better use DTIC in the treatment of those neoplasms for which this agent has demonstrated activity.

In the present study, we characterized the human in vitro metabolism of DTIC by delineating the specific P450 isoforms responsible for MTIC formation in the liver. This data provides a more complete understanding of DTIC metabolic activation and of the basis for differences in DTIC activity in animal models and humans. This data may also contribute to improvements in the therapeutic effects of DTIC by optimizing metabolic activation in patients.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
DTIC and paclitaxel were obtained from the Pharmaceutical Resources Branch, National Cancer Institute (Bethesda, MD). SKF525A was a generous gift from SmithKline Beecham (Philadelphia, PA). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADP were obtained from Boehringer Mannheim, (Indianapolis, IN). AIC, 6-methyl-nicotinamide, metyrapone, {alpha}-naphthoflavone, coumarin, tolbutamide, quinidine, disulfiram, erythromycin, quercetin, chlorzoxazone, phenacetin, sulfaphenazole, and caffeine were obtained from Sigma Chemical Co. (St. Louis, MO). All of the other reagents and HPLC solvents were the highest grade available and were used as received (e.g., heptane sulfonic acid, triethylamine, phosphoric acid, methanol, DMSO, hydrochloric acid, KH2PO4, K2HPO4, Tris, and MgCl2).

Human Liver Microsomal Fractions.
Human liver microsomal fractions were provided by Jerry M. Collins (Center for Drug Evaluation and Research, United States Food and Drug Administration, Rockville, MD). Human liver samples (HL5, HL6, HL7, and HL8), medically unsuitable for liver transplantation, were acquired under the auspices of the Washington Regional Transplant Consortium (Washington, DC). A description of the preparation and characterization of the liver microsomal fractions has been published previously (14) .

Incubation Conditions.
Microsomal suspensions were incubated in amber glass vials maintained at 37°C in a shaker bath. Each 1-ml incubation mixture contained human liver microsomes (1 mg of protein), NADP+ (0.4 mM), glucose-6-phosphate (25 mM), glucose-6-phosphate dehydrogenase (0.7 units/ml), magnesium chloride (5 mM), and Tris (50 mM) or potassium phosphate (100 mM) buffer adjusted to pH 7.4. Control incubation mixtures contained boiled microsomes or active microsomes with a nitrogen or CO/O2 atmosphere. The incubation mixtures were preincubated for 2 min before the initiation of the reaction on the addition of 500 µM DTIC. At the end of the incubation period, reactions were terminated by the addition of 2 ml of ice-cold methanol that contained 50 ng 6-methylnicotinamide as an internal standard for HPLC analysis. The aqueous methanol supernatants obtained after centrifugation (10,000 x g for 2 min) were concentrated to dryness under vacuum and reconstituted in HPLC mobile phase for analysis. Chemical inhibitors were dissolved in various solvents (water, methanol, or DMSO). Appropriate controls containing drug vehicles only were also performed in parallel incubations.

Correlation of DTIC Metabolism with Marker Activities of Selected Human P450 Forms.
A Hepatoscreen Test kit that contained 10 human liver microsome preparations was obtained from Human Biologicals, Inc. (Phoenix, AZ). Microsomal suspensions were prepared in the reaction buffer to achieve a final protein concentration of 1 mg/ml and were incubated with DTIC (500 µM) for a 30-min reaction period as described above.

Metabolite Formation by cDNA-expressed Human P450 Enzymes.
Microsomal suspensions from the B-lymphoblastoid cell line AHH-1 TK+/- expressing cDNA constructs for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9a, CYP2C9c, CYP2E1, and CYP3A4 were obtained from Gentest Corporation (Woburn, MA). After dilution with the reaction buffer to achieve a final protein concentration of 1 mg/ml, microsomal suspensions were incubated with DTIC (500 µM) for reaction periods of 30 min and 120 min. Control reactions included microsomal preparation from cells that contained the expression vector alone. Because documentation provided by Gentest noted that common organic solvents such as methanol, ethanol, DMSO, and so forth, inhibited some P450 forms, DTIC stock solutions were prepared in 0.01 N HCl.

In experiments to determine the kinetics of DTIC N-demethylation by CYP1A1, CYP1A2, and CYP2E1, each 100-µl incubation mixture contained DTIC (200–2000 µM) human liver microsomes (0.05 mg protein), and the cofactor mixture described in "Incubation Conditions." Reactions were terminated after 45-min incubation.

Immunoinhibition.
Antihuman CYP1A1 and CYP1A2 antiserum (Gentest Corporation, Woburn, MA) was preincubated with human liver microsomes on ice for 30 min before adding reaction buffer and DTIC (500 µM). Incubations were carried out for 30 min as described above.

Sample Preparation and HPLC Analysis.
After termination of the reactions by protein precipitation with methanol, the supernatants obtained by centrifugation were dried under vacuum, reconstituted in mobile phase, and kept at room temperature for no less than 20 min before HPLC to ensure conversion of HMMTIC and MTIC to AIC.

Separations were achieved by a column-switching technique in which an Apex I octadecyl precolumn (Jones Chromatography, 3 cm x 4.6 mm; 5 µm) was installed in a 10-port Valco switching valve in place of a sample loop and an Apex I (Jones Chromatography, Littleton, CO) octadecyl analytical column (25 cm x 4.6 mm; 5 µm) was installed between the switching valve solvent exit port and the UV absorbance detector. This configuration permitted rapid analysis because late-eluting peaks could be passed to waste before elution onto the analytical column. The mobile phase consisted of 90% 10 mM heptane sulfonic acid, 50 mM phosphoric acid (pH 2.0) with 0.1% triethylamine and 10% methanol. The flow rate, injection volume, and UV absorbance wavelength were 1 ml/min, 40 µl, and 280 nm, respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metabolism of DTIC by Human Liver Microsomes.
N-Demethylation of DTIC was monitored by the formation of AIC, the decomposition product of the initial oxidative metabolite HMMTIC. Reverse-phase HPLC with a mobile phase containing heptane sulfonic acid permitted separation of AIC, the internal standard 6-methyl-nicotinamide and other components of the incubation mixture. A column-switching technique prevented nonpolar materials from eluting onto the analytical column, eliminated interferences, and allowed reduced run times.

The P450-dependent N-demethylation of DTIC and concomitant formation of AIC by human liver microsomes was confirmed by several observations. AIC formation was dependent on the presence of NADPH and oxygen in the incubation mixtures, inhibited by replacing the atmosphere in the reaction vessel with N2 and absent from incubation mixtures prepared with boiled microsomal suspensions (data not shown).

The inhibition of DTIC N-demethylation in HL8 liver microsomes by chemical inhibitors of P450 enzymes is summarized in Fig. 2Citation . The most potent inhibitors of AIC formation were {alpha}-naphthoflavone, quercetin, phenacetin, chlorzoxazone, and disulfiram, which suggests CYP1A, CYP2E1, and CYP3A enzymes contributed to DTIC metabolism. {alpha}-Naphthoflavone, quercetin, phenacetin, and chlorzoxazone were also potent inhibitors of DTIC metabolism by three other liver microsome preparations (data not shown). With the exception of coumarin (CYP2A), other P450 inhibitors-selective (erythromycin, tolbutamide, sulfaphenazole, and quinidine) did not reduce DTIC metabolism (data not shown). Concentration-dependent inhibition by quercetin, chlorzoxazone, and disulfiram was observed over the range 20–200 µM, whereas inhibition by {alpha}-naphthoflavone was essentially complete (>85%) at concentrations above 0.2 µM(Fig. 3A)Citation . The predominant role of CYP1A2 in human liver microsomes was confirmed by incubation with antihuman CYP1A2 antibody, which inhibited DTIC metabolism in four human liver microsome by greater than 75% (Fig. 3B)Citation .



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Inhibition of DTIC metabolism by selective chemical inhibitors of various P450 subfamilies. DTIC was incubated with human liver microsomes in the presence of 20 µM (open bar) and 200 µM (filled bar) of the inhibitor. Error bars, the SD of the mean values obtained from three experiments.

 


View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Inhibition of DTIC metabolism in human liver microsomes. A, HL8 microsomes were incubated with DTIC in the presence of the chemical inhibitors {alpha}-naphthoflavone (•), disulfiram ({blacktriangledown}), quercetin ({blacksquare}), and chlorzoxazone ({blacktriangleup}). B, HL5 (•), HL6 ({blacktriangledown}), HL7 ({blacksquare}) and HL8 ({blacktriangleup}) microsomes were incubated with DTIC after 30 min preincubation with antihuman CYP1A2 antiserum.

 
Correlation of DTIC Metabolism with Marker Activities of Selected Human P450 Forms.
DTIC metabolism was studied in a panel of 10 human liver microsomes with defined catalytic activities for major P450-selective substrates. The median (n = 10) rate of NADPH-dependent AIC formation from DTIC by human liver microsomes was 76 pmol/min/mg protein (range, 34–463 pmol/min/mg protein). Among the nine marker activities examined (Table 1)Citation , the highest correlation coefficients (r > 0.94) were found for the relationship between AIC formation and the CYP1A2 markers caffeine N3-demethylation and 7-ethoxyresorufin O-dealkylation (Fig. 4)Citation . The correlation coefficients for the relationships between AIC formation and marker activities representing other P450s (CYP2A, CYP2C, CYP2D, CYP2E, CYP3A, and CYP4A) were much lower (r < 0.40). Incubation of DTIC with microsomal suspensions in the presence of 2 µM {alpha}-naphthoflavone reduced DTIC metabolism by 38–96% and abolished the correlations with the CYP1A2 marker activities. This residual DTIC metabolism was correlated with chlorzoxazone 6-hydroxylation, tolbutamide methylhydroxylation, and testosterone 6ß-hydroxylation (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1 Correlation of DTIC N-demethylation with catalytic activities of human P450-selective substrates

 


View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Correlation of DTIC metabolism with (A) 7-ethoxyresorufin O-dealkylation (r = 0.946), and (B) caffeine N3-demethylation (r = 0.977) in a panel of 10 human liver microsome preparations. Enzyme activity data are expressed as pmol product formed/mg protein/min. Values, the mean of three experiments.

 
Metabolism by cDNA-expressed human P450s.
Recombinant human P450 isoforms were used to verify individual P450s that catalyze DTIC metabolism. DTIC metabolism was catalyzed by cDNA-expressed CYP1A1, CYP1A2, and CYP2E1 but not by CYP2C9 or CYP3A4. The amount of product formed was linear with time, and the catalytic activity of CYP1A1 (3.62 ± 0.75 pmol AIC formed/pmol P450/min) was 3-fold greater than that of CYP1A2 (1.26 ± 0.24 pmol AIC formed/pmol P450/min) and 16-fold greater than that of CYP2E1 (0.22 ± 0.04 pmol AIC formed/pmol P450/min). {alpha}-Naphthoflavone, quercetin, chlorzoxazone, and phenacetin-inhibited CYP1A1- and CYP1A2-catalyzed DTIC metabolism. Chlorzoxazone and phenacetin were selective for CYP1A2-catalyzed DTIC metabolism (data not shown).

The kinetics of DTIC N-demethylation were defined with individual cDNA-expressed enzymes because saturable enzyme kinetics were not observed in human microsomes. The Km and Vmax values for CYP1A1 were 595 ± 111 µM and 10.2 ± 2.1 pmol product/pmol P450/min, respectively. The Km and Vmax values for CYP1A2 were 659 ± 88 µM and 13.8 ± 2.4 pmol product/pmol P450/min, respectively. Saturable kinetics were not observed for cDNA-expressed CYP2E1 even when the enzyme concentration was reduced 4-fold from 0.5 to 0.125 mg/ml microsomal protein. Analysis of the CYP2E1 kinetic data over the range studied suggests the Km values exceeded 2.8 mM. Higher concentrations were not examined because of the limited aqueous solubility of DTIC.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that human CYP1A1, CYP1A2, and CYP2E1 catalyzed in vitro metabolism of DTIC, based on the collective results of chemical- and immuno-inhibition studies, correlations with marker substrates of P450 isoform activities in a human liver panel, and metabolism by cDNA expression systems. The combined data from several in vitro approaches were used to infer the predominant catalytic activity because the results of individual approaches can lead to erroneous conclusions.

The principle P450 isoform responsible for the in vitro metabolism of DTIC by human liver microsomes is CYP1A2. With the exception of coumarin, each of the chemicals that inhibited DTIC metabolism were inhibitors of CYP1A2. {alpha}-Naphthoflavone (15) and phenacetin (16) are selective inhibitors of CYP1A subfamily isozymes. The CYP3A subfamily inhibitor quercetin (17) and CYP2E1 inhibitors disulfiram (18) and chlorzoxazone (19) also inhibit CYP1A subfamily isozymes (15 , 19 , 20) . A complementary study of the DTIC metabolism in a panel of human liver microsomes found a correlation only with catalytic activities for CYP1A2 substrates ethoxyresorufin and caffeine. The key role of CYP1A2 in human liver microsomal metabolism of DTIC was confirmed by nearly complete inhibition by an antihuman CYP1A2 antibody.

In contrast to CYP1A2, CYP2E1 most likely plays a secondary role based on lower affinity and lower expression in liver tissue. DTIC metabolism in a panel of human liver microsomes was correlated with the catalytic activity for CYP2E1 only when the CYP1A2 catalytic activity was inhibited by {alpha}-naphthoflavone. In addition, CYP2E1 (7%) accounts for a lower fraction of the total liver cytochrome P450 content than CYP1A2 (13%; Ref. 21 ). Although residual DTIC metabolism in {alpha}-naphthoflavone containing incubations was also correlated with testosterone 6ß-hydroxylation and tolbutamide methylhydroxylation, it is unlikely that CYP3A4 and CYP2C9, respectively, are involved in DTIC metabolism. Of the CYP3A4-selective inhibitors examined, only quercetin, which is cross-reactive with CYP1A2, reduced DTIC metabolism in human liver microsomes. This inhibitor also reduced cDNA-expressed CYP1A1- and CYP1A2-catalyzed DTIC metabolism. The CYP2C9-selective inhibitor tolbutamide did not inhibit DTIC metabolism. Finally, cDNA-expressed CYP2C9 and CYP3A4 did not metabolize DTIC.

The catalytic activity of CYP1A1 is interesting because this enzyme is found in extrahepatic tissue. Using highly selective antibodies against CYP1A1 and CYP1A2, we confirmed observations of other investigators (6 , 22, 23, 24) that CYP1A1 is not found in the liver to any appreciable extent.5 Although detectable levels of CYP1A1 mRNA have been found in human liver (22) , protein content is low or undetectable even in individuals exposed to substances that induce CYP1A1 in other tissues (6 , 23 , 24) . Because CYP1A1 is also expressed in some tumors, activation of DTIC to DNA-methylating metabolites may occur within tumors.

There is substantial variability in P450 expression among individuals because of genetic, physiological, and environmental factors (23) . A 40-fold range in CYP1A2 protein concentration and mRNA expression has been reported in human liver microsome panels (24) . The DTIC catalytic activity in the liver panel used in these investigations varied over a 13-fold range. Consequently, the variability of DTIC antitumor activity and toxicity may be associated with the variability of CYP1A2 expression. The potential clinical impact of DTIC activation by CYP1A2 may be investigated by exploring the relationship between DTIC pharmacokinetics and CYP1A2 phenotype, using caffeine as a probe drug (25) .

The antitumor activity and toxicity of DTIC may be altered by concomitant medications. Because DTIC has limited activity as a single agent, it has been combined with other antitumor agents to improve response (26, 27, 28) . Drug interactions between DTIC and drugs most often used in combination with DTIC, including doxorubicin, cyclophosphamide, ifosphamide, and vincristine (29 , 30) , are unlikely because none are known inhibitors or inducers of CYP1A2 or CYP2E1. An interaction between DTIC and O6-benzylguanine, an inhibitor of the DNA repair enzyme AGT (31) , is possible because O6-benzylguanine is also a CYP1A2 substrate (32) . Anticonvulsants that induce CYP3A4 and are commonly prescribed in the treatment of brain tumors would not be expected to exhibit interactions with DTIC (12) . The CYP1A inducer omeprazole (33 , 34) and the CYP2E1 inducer isoniazid (6) may induce the DTIC metabolism and potentiate its activity or toxicity.

It is unlikely that the restricted spectrum of DTIC activity against human tumors compared with rodent tumors is due to lower metabolic activation in humans. In fact, DTIC metabolism by humans is most likely equivalent, if not greater than the metabolism by rodents. The kinetics of DTIC metabolism for mouse liver microsomes (Km = 0.25–1.33 mM and Vmax = 29 pmol/min/mg protein; Ref. 2 ) are equivalent to those for CYP1A2, which was the high-affinity enzyme in human liver microsomes (Km = 0.6 mM). Recently, Yamagata et al. (35) reported that CYP1A subfamily enzymes contribute to DTIC metabolic activation by rat liver microsomes. CYP1A subfamily enzymes comprise 13% of the total amount of P450 in humans but <3% of the P450 in the rat (9) .

In summary, we identified three human P450s that catalyze the metabolism of DTIC. The principle hepatic P450 that catalyzes DTIC metabolism is CYP1A2, but CYP2E1 may participate under circumstances in which CYP1A2 expression is low. CYP1A1, primarily an extrahepatic P450, also catalyzes DTIC metabolism and may be important to the activation of DTIC at its site of action. Differences in P450 expression may account for the variability in DTIC metabolism in patients.


   ACKNOWLEDGMENTS
 
We thank Dr. Scott H. Kaufmann for careful review of the manuscript and Wanda Rhodes for preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Mayo Cancer Center Grant CA15083. Back

2 To whom requests for reprints should be addressed, at Mayo Clinic, Department of Oncology, Division of Developmental Oncology Research, 200 First Street S.W., Rochester, MN 55905. Phone: (507) 284-4303; Fax: (507) 284-3906. Back

3 Current address: Department of Chemistry, University of Puerto Rico, Mayaguez Campus, P. O. Box 5000, Mayaguez, PR 00681-5000. Back

4 The abbreviations used are: DTIC, dacarbazine; MTIC, 5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide; HMMTIC, 5-[3-hydroxy-methyl-3-methyl-triazen-l-yl]-imidazole-4-carboxamide; AIC, aminoimidazole carboxamide; HPLC, high-pressure liquid chromatography; P450, cytochrome P450. Back

5 Unpublished data. Back

Received 10/14/99; revised 5/12/99; accepted 5/14/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Legha S. S. Current therapy for malignant melanoma. Semin. Oncol., 16: 34-44, 1989.
  2. Hill D. L. Microsomal metabolism of trizenylimidazoles. Cancer Res., 35: 3106-3110, 1975.[Abstract/Free Full Text]
  3. Skibba J. L., Beal D. D., Ramirez G., Bryan G. T. N-Demethylation of the antineoplastic agent 4(5)-3,3-dimethyl-1-trizeno)-imidazole-5(4)-carboxamide by rats and man. Cancer Res., 30: 147-150, 1970.[Abstract/Free Full Text]
  4. Meer L., Janzer R. C., Kleihues P., Kolar G. F. In vivo metabolism and reaction with DNA of the cytostatic agent, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). Biochem. Pharmacol., 35: 3243-3247, 1986.[Medline]
  5. Nelson D. R., Koymans L., Kamataki T., Stegeman J. J., Feyereisen R., Waxman D. J., Waterman M. R., Gotoh O., Coon M. J., Estrabrook R. W., Gunsalus I. C., Nebert D. W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 6: 1-42, 1996.[Medline]
  6. Wrighton S. A., Vandenbranden M., Stevens J. C., Shipley L. A., Ring B. J., Rettie A. E., Cashman J. R. In vitro methods for assessing human hepatic drug metabolism: their use in drug development. Drug Metab. Rev., 25: 453-484, 1993.[Medline]
  7. Breithaupt H., Dammann A., Aigner K. Pharmacokinetics of dacarbazine (DTIC) and its metablite 5-aminoimidazole-4-carboxamide (AIC) following different dose schedules. Cancer Chemother. Pharmacol., 9: 103-109, 1982.[Medline]
  8. Buesa J. M., Urrechaga E. Clinical pharmacokinetics of high-dose DTIC. Cancer Chemother. Pharmacol., 28: 475-479, 1991.[Medline]
  9. Nedelcheva V., Gut I. P450 in the rat and man: methods of investigation, substrate specificities and relevance to cancer. Xenobiotica, 24: 1151-1175, 1994.[Medline]
  10. Rutty C. J., Newell D. R., Vincent R. B., Abel G., Goddard P. M., Harland S. J., Calvert A. H. The species dependent pharmacokinetics of DTIC. Br. J. Cancer, 48: 140 1983.
  11. Bleehen N. M., Newlands E. S., Lee S. M., Thatcher N., Selby P., Calvert A. H., Rustin G. J. S., Brampton M., Stevens M. F. G. Cancer Research Campaign Phase II trial of temozolomide in metastatic melanoma. J. Clin. Oncol., 13: 910-913, 1995.[Abstract]
  12. O’Reilly S. M., Newlands E. S., Glaser M. G., Brampton M., Rice-Edwards J. M., Illingworth R. D., Richards P. G., Kennard C., Colquhoun I. R., Lewis P., Stevens M. F. G. Temozolomide: a new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours. Eur. J. Cancer, 29A: 940-942, 1993.
  13. Stevens M. F. G., Hickman J. A., Langdon S. P., Chubb D., Vickers L., Stone R., Baig G., Goddard C., Gibson N. W., Slack J. A., Newton C., Lunt E., Fizames C., Lavelle F. Antitumor activity and pharmacokinetics in mice of 8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (CCRG 81045; M & B 39831), a novel drug with potential as an alternative to dacarbazine. Cancer Res., 47: 5846-5852, 1987.[Abstract/Free Full Text]
  14. Harris J. W., Rahman A., Kim B.-R., Guengerich F. P., Collins J. M. Metabolism of Taxol by human hepatic microsomes and liver slices: participation of cytochrome P450 3A4 and an unknown P450 enzyme. Cancer Res., 54: 4026-4035, 1994.[Abstract/Free Full Text]
  15. Chang T. K. H., Gonzalez F. J., Waxman D. J. Evaluation of triacetyloleandomycin, {alpha}-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Biochem. Biophys., 311: 437-442, 1994.[Medline]
  16. Distlerath L. M., Reilly P. E., Martin M. V., Davis G. G., Wilkinson G. R., Guengerich F. P. Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydrxoylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 260: 9057-9067, 1985.[Abstract/Free Full Text]
  17. Miniscalco A., Lundahl J., Regardh C. G., Edgar B., Eriksson U. G. Inhibition of dihydropyridine metabolism in rat and human liver microsomes by flavonoids found in grapefruit juice. J. Pharm. Exp. Ther., 261: 1195-1199, 1992.[Abstract/Free Full Text]
  18. Guengerich F. P., Kim D. H., Iwasaki M. Role of human cytochrome P450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol., 4: 168-179, 1991.[Medline]
  19. Ono S., Hatanaka T., Hotta H., Tsutsui M., Satoh T., Gonzalez F. J. Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics, 5: 143-150, 1995.[Medline]
  20. Le Bon A.-M., Siess M.-H., Suschetet M. Inhibition of microsome-mediated binding of benzo[{alpha}]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats. Chem.-Biol. Interact., 83: 65-71, 1992.[Medline]
  21. Shimada T., Yamazaki H., Mimura M., Inui Y., Guengerich F. P. Interindividual variations in human liver cytochrome P-450 enzymes involved in th oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharm. Exp. Ther., 270: 414-423, 1994.[Abstract/Free Full Text]
  22. Schweikl H., Taylor J. A., Kitareewan S., Linko P., Nagorney D., Goldstein J. A. Expression of CYP1A1 and CYP1A2 genes in human liver. Pharmacogenetics, 3: 239-249, 1993.[Medline]
  23. Murray B. P., Edwards R. J., Murray S., Singleton A. M., Davies D. S., Boobis A. R. Human hepatic CYP1A1 and CYP1A2 content, determined with specific anti-peptide antibodies, correlates with the mutagenic activation of PhIP. Carcinogenesis (Lond.), 14: 585-592, 1993.[Abstract/Free Full Text]
  24. Drahushuk A. T., McGarrigle B. P., Larsen K. E., Stegeman J. J., Olson J. R. Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture. Carcinogenesis (Lond.), 19: 1361-1368, 1998.[Abstract/Free Full Text]
  25. Fuhr U., Rost K. L., Engelhardt R., Sachs M., Liermann D., Belloc C., Beaune P., Janezic S., Grant D., Meyer U. A., Staib A. H. Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations. Pharmacogenetics, 6: 159-176, 1996.[Medline]
  26. Buzaid A. C., Legha S. S. Combination of chemotherapy with interleukin-2 and interferon-{alpha} for the treatment of advanced melanoma. Semin. Oncol., 21: 23-28, 1994.[Medline]
  27. Valle A. A., Kraybill W. G. Management of soft tissue sarcomas of the extremity in adults. J. Surg. Oncol., 63: 271-279, 1996.[Medline]
  28. Yuen A. R., Horning S. J. Hodgkin’s disease: management of first relapse. Oncology, 10: 233-240, 1996.[Medline]
  29. LeBlanc G. A., Waxman D. J. Interaction of anticancer drugs with hepatic monooxygenase enzymes. Drug Metab. Rev., 20: 395-439, 1989.[Medline]
  30. Kivisto K. T., Kroemer H. K., Eichelbaum M. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions. Br. J. Clin. Pharmacol., 40: 523-530, 1995.[Medline]
  31. Gerson S. L., Berger N. A., Arce C., Petzold S. J., Willson J. K. V. Modulation of nitrosourea resistance in human colon cancer by O6-methylguanine. Biochem. Pharmacol., 43: 1101-1107, 1992.[Medline]
  32. Roy S. K., Korzekwa K. R., Gonzalez F. J., Moschel R. C., Dolan M. E. Human liver oxidative metabolism of O6-benzylguanine. Biochem. Pharmacol., 50: 1385-1389, 1995.[Medline]
  33. Rost K. L., Brosicke H., Brockmoller J., Scheffler M., Helge H., Roots I. Increase of cytochrome P450IA2 activity by omeprazole: evidence by the 13C-[N-3-methyl]-caffeine breath test in poor and extensive metabolizers of S-mephenytoin. Clin. Pharmacol. Ther., 52: 170-180, 1992.[Medline]
  34. Rost K. L., Roots I. Accelerated caffeine metabolism after omeprazole treatment is indicated by urinary metabolite ratios: coincidence with plasma clearance and breath test. Clin. Pharmacol. Ther., 55: 402-411, 1994.[Medline]
  35. Yamagata S.-I., Ohmori S., Suzuki N., Yoshino M., Hino M., Ishii I., Kitada M. Metabolism of dacarbazine by rat liver microsomes contribution of CYP1A enzymes to dacarbazine N-demethylation. Drug Metab. Dispos., 26: 379-382, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J AndrolHome page
R. Obligacion, M. Murray, and I. Ramzan
Drug-Metabolizing Enzymes and Transporters: Expression in the Human Prostate and Roles in Prostate Drug Disposition
J Androl, March 1, 2006; 27(2): 138 - 150.
[Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
M. Rooseboom, J. N. M. Commandeur, and N. P. E. Vermeulen
Enzyme-Catalyzed Activation of Anticancer Prodrugs
Pharmacol. Rev., March 1, 2004; 56(1): 53 - 102.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. R. Bacon, G. Williamson, R. C. Garner, G. Lappin, S. Langouet, and Y. Bao
Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes
Carcinogenesis, December 1, 2003; 24(12): 1903 - 1911.
[Abstract] [Full Text] [PDF]

Home page
 MutagenesisHome page
I.-D. Adler, U. Kliesch, I. Jentsch, and M.R. Speicher
Induction of chromosomal aberrations by dacarbazine in somatic and germinal cells of mice
Mutagenesis, September 1, 2002; 17(5): 383 - 389.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
L. Long and M. E. Dolan
Role of Cytochrome P450 Isoenzymes in Metabolism of O6-Benzylguanine: Implications for Dacarbazine Activation
Clin. Cancer Res., December 1, 2001; 7(12): 4239 - 4244.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
N. J. Toft, O. J. Sansom, R. A. Brookes, M. J. Arends, M. Wood, G. P. Margison, D. J. Winton, and A. R. Clarke
In vivo administration of O6-benzylguanine does not influence apoptosis or mutation frequency following DNA damage in the murine intestine, but does inhibit P450-dependent activation of dacarbazine
Carcinogenesis, April 1, 2000; 21(4): 593 - 598.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reid, J. M.
Right arrow Articles by Ames, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reid, J. M.
Right arrow Articles by Ames, M. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online