Differential effects of ibuprofen,indomethacin, and meclofenamate on prostaglandin endoperoxide H2 metabolism

Summary Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A₂ synthase activity in addition to inhibiting cyclooxygenae activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI₂) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E₂ isomerase activities. When [1-¹⁴C] PG endoperoxide H₂ (PGH₂) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI₂ synthase, TXA₂ synthase of GSH-dependent PGE₂ isomerase, whereas indomethacin selectively inhibited PGI₂ synthase activity at 5 x 10^–4 M and 10^–3 M. Meclofenamate selectively inhibited TXA₂ synthase activity at 5 x 10^–4 M and 10^–3 M. At concentrations of 5 x 10^–3 M, this selectivity was not oberved, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF₁ and TXB₂. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH₂ metabolism. This action may affect the physiologic consequences of the exchange of PGH₂ between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI₂ synthase activity in vitro.     

Prostaglandin synthase-catalyzed metabolic activation of some aromatic amines to genotoxic products

Cultured human fibroblasts were incubated with different aromatic amines in the presence of different activation systems and the induction of strand breaks in fibroblast DNA was studied. In the presence of ram seminal vesicle microsomes and arachidonic acid, DNA strand breaks were induced by 2-naphthylamine, 2,4-diaminotoluene and 4-methoxy-m-phenylenediamine. This effect was decreased when the prostaglandin synthase of the ram seminal vesicle microsomes was inhibited. The data suggest that metabolic activation catalyzed by prostaglandin synthase may be of importance in the formation of genotoxic products by certain urinary tract carcinogens.     

Quantum chemical studies of the metabolism of polycyclic aromatic amines and the stabilities and electrophilicities of their arylnitrenium ions in relation to their mutagenic/carcinogenic potencies.

Electronic parameters related to the cytochrome P450-catalyzed reactions of eight polycyclic aromatic amines have been calculated using all valence electron semiempirical molecular orbital methods. The reactions considered lead to the presumably carcinogenic arylnitrenium ions and to the competing hydroxylation and epoxidation products. The stabilities of the arylnitrenium ions relative to the N-hydroxylamines and their sulfate esters were also calculated, together with electrophilic reactivity parameters of the ions. The resulting parameters were used to predict major metabolites of the parent compounds and also to correlate with observed mutagenic activities of the four pairs of polycyclic aromatic amines studied. The major factor in determining mutagenic potencies of parent compounds appears to be the extent of N-hydroxylation and competing ring oxidations, as well as the electrophilic properties of the arylnitrenium ions.     

Fungal oxidation of 3-methylcholanthrene: Formation of proximate carcinogenic metabolites of 3-methylcholanthrene

The filamentous fungus, Cunninghamella elegans, was found to metabolize the potent carcinogen, 3-methylcholanthrene (3-MC) to 1-hydroxy-3-MC, 2-hydroxy-3-MC, 1-keto-3-MC, 2-keto-3-MC and trans-9,10-dihydrodiols of 1-hydroxy-3-MC. In addition several unidentified derivatives of 3-MC were found. The metabolites formed were separated by high pressure liquid chromatography (HPLC) and identified by comparison of retention times, absorbance, fluorescence and mass spectra with those of synthetic standards. Incubation of (±)-1-hydroxy-3-MC and (±)-2-hydroxy-3-MC with cells of C. elegans indicated that 1-hydroxy-3-MC is metabolized to form diasteromerically related trans-9,10-dihydrodiols of 1-hydroxy-3-MC. Experiments with 3-[¹⁴C]MC showed that over a 48-h period, 8.7% of the hydrocarbon was oxidized to organic solvent-soluble metabolic products. Most of the metabolites were polar products, some of which co-chromatographed with trans-9,10-dihydrodiols of 1-hydroxy-3-MC. The results show that C. elegans has the ability to oxidize 3-MC to metabolites that have been implicated as proximate carcinogenic forms of 3-MC in higher organisms.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture

Investigations on the metabolism of ³H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-dibenzo[c,g]carbazole metabolism by microsomal enzymes from mouse and rat liver

The metabolism of dibenzo[c,g]carbazole (DBC), was studied in vitro using microsomal fractions of mouse and rat liver from animals, which were treated with 3-methylcholanthrene (MC). The separation of extractable metabolites by high pressure liquid chromatography (HPLC) and thinlayer chromatography (TLC) as well as identification of most of them by nuclear magnetic resonance, mass spectrometry and comparison with synthetically obtained products are described. The microsomes of both species produced the same twelve compounds of which the following have been identified: five monohydroxylated derivatives (phenols), the product of further oxidation of one of them, and a dihydrodiol. The 5-OH-DBC (60% including its spontaneously-formed dimer) and the 3-OH-DBC (14%) are the main metabolites. Three minor metabolites cochromatographed with synthetically prepared 2-OH-DBC, 4-OH-DBC and 6-OH-DBC. The dihydrodiol detectable in small quantity (4–6%) was tentatively identified as 3,4-dihydroxy-3,4-dihydro-DBC by the sensitivity of its formation to very low concentrations of the inhibitor of microsomal epoxide hydrolase, 1,1,1-trichloropropene oxide, by its molecular ion and major fragment in mass spectrometry and by its dehydration product 3-OH-DBC. No other dihydrodiols were detected. The qualitative and quantitative effects of various modulators of metabolism (enzyme inhibitors, apparently homogeneous epoxide hydrolase, glutathione, supernatant fraction) were investigated. The results are discussed with respect to possible ultimate carcinogens.     

The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).     

Conformations of adducts and kinetics of binding to DNA of the optically pure enantiomers of anti-benzo(a)pyrene diol epoxide

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (-) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (-) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.     

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.     

Metabolism of methyl n-amyl ketone (2-heptanone) and its binding to DNA of rat liver in vivo and in vitro

Methyl n-amyl ketone (2-heptanone), a reported metabolite of 2-ethylhexanol which in turn is a primary metabolite of plasticizers such as di-(2-ethylhexyl) phthalate, is metabolized in male Fischer 344 rats to CO2, acetate and a variety of compounds that could be either anabolic or catabolic or a combination of the two. A significant percentage of the radioactivity given orally (gavage) as [2-14C]-2 heptanone, at least 10%, was not excreted from the body in 48 h. Radioactivity was incorporated into liver protein in the form of three unidentified products as well as [14C]arginine, and into DNA both as 14C-labeled normal nucleosides (50-75%) and as presently unidentified hydrophobic materials (25-50%). Urea and cholesterol were significantly labeled, indicative of anabolic reutilization of [2-14C]-2-heptanone breakdown products. The 2-heptanone also bound to DNA spontaneously in vitro, to the extent of 400 pmol/mg DNA.     

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.     

Metabolism and nucleic acid binding of 7-fluoro-2-acetamidofluorene in rats: oxidative defluorination and apparent dissociation from hepatocarcinogenesis of 8-(N-arylamide) guanine adducts on DNA

The significance in hepatocarcinogenesis of various arylamine/amide adducts with nucleic acid was investigated by the use of comparison studies on several different parameters. Female Fischer and Sprague-Dawley rats are comparably sensitive to hepatocarcinogenesis by 2-acetamidofluorene (AAF), while male rats are more sensitive. 7-Fluoro-AAF is more carcinogenic in Sprague-Dawley rats than is AAF, but is strikingly so toward the liver of the female rat. Based on these observations, binding of both compounds to liver nucleic acids was determined for male and female Fischer rats at 1 and 3 days after a single injection of carcinogen, and in female Sprague-Dawley rats from 1 to 28 days after a single injection. As shown by others, no 8-(N-2-fluorenylacetamido)guanine adduct could be found in RNA or DNA of female Sprague-Dawley rats treated with AAF (nor was the corresponding 7-fluoro derivative detectable). These adducts were present, however, in comparable amounts in both male and female Fischer rats. The binding of 7-fluoro-AAF derivatives was higher than that of AAF derivatives in female Sprague-Dawley rats. Feeding of either AAF or 7-fluoro-AAF to Sprague-Dawley rats for 4 weeks before a single injection of [3H]7-fluoro-AAF resulted in reduction of the 8-(N-2-(7-fluoro)fluorenylacetamido)guanine adduct in males to undetectable levels in DNA and to 10% of control level in RNA. Non-acetylated adducts were increased in males, but decreased in females by AAF prefeeding; 7-fluoro-AAF prefeeding resulted in little change in adduct formation in females and in a major increase in non-acetylated adducts in males. AAF adducts disappeared from DNA more rapidly than did 7-fluoro-AAF adducts. Assay of the urinary metabolites from the animals in the prefeeding experiment showed that all compounds fed (including the non-hepatocarcinogens 4-acetamidobiphenyl and 2-acetamidophenanthrene) increased the proportion of N-hydroxy-7-fluoro-AAF among the metabolites. Defluorination of 7-fluoro-AAF to 7-hydroxy-AAF was also demonstrated and the ratio of 7-hydroxy-AAF to 5-hydroxy-7-fluoro-AAF was comparable to that observed for 7-hydroxy-AAF/5-hydroxy-AAF and AAF itself, suggesting that fluoro substitution does not increase activity by preventing detoxication.     

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

High-density lipoproteins decrease both binding of a polynuclear aromatic hydrocarbon carcinogen to DNA and carcinogen-initiated cell transformation

Lipophilic carcinogens partition into the three major classes of lipoproteins potentially present in serum used as a medium supplement for cell culture. Different serum lots sequester differing quantities of the procarcinogen benzo[a]pyrene, dependent on the serum lipoprotein concentrations. In the presence of high-density lipoproteins a mutagenic benzo[a]pyrene metabolite was bound to cellular DNA at decreased levels when compared to cells exposed to the mutagen in the absence of high-density lipoproteins. Fetal calf serum with low levels of lipoproteins, specifically, high-density lipoproteins, is associated with efficient methylcholanthrene-initiated transformation of C3H/10T1/2 cells, while calf serum with a significant concentration of high-density lipoproteins requires up to a 500% increase in methylcholanthrene concentration to achieve similar levels of transformation in this mouse embryo cell line. When concentrated serum lipoproteins or purified HDL were added to fetal calf serum containing MCA at μg/ ml, the C3H/10T1/2 transformation frequency was decreased compared to the transformation frequency achieved in the presence of 1 μg/ml of MCA in fetal calf serum without supplementation. The results suggest that high-density lipoprotein partitioning of lipophilic polynuclear aromatic hydrocarbon mutagens from the cell culture medium may effectively reduce the concentration of carcinogen available for interaction with cellular DNA in vitro, which, in turn, may be associated with decreased carcinogen-induced transformation of cells.     

Species difference in the metabolic activation of phenacetin by rat and hamster liver microsomes

Phenacetin is mutagenic in Salmonella typhimurium TA 100 when liver 9,000 X g supernatant fractions from PCB-treated hamsters instead of rats are used. A mechanism of the species difference in phenacetin mutagenicity was investigated. By high-performance liquid chromatography analysis, it was found that phenacetin is activated to direct-acting mutagens through N-hydroxylation and deacetylation by hamster liver microsomes. Although no significant species difference was observed in N-hydroxylation, rates of deacetylation were 9 to 150 times higher in hamsters than in rats. The results indicate that the marked species difference in phenacetin mutagenicity is due to the difference in deacetylation activity between rat and hamster liver microsomes.