Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide.

ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.

Synthetic oligonucleotides: the development of antisense therapeutics.

Antisense therapeutics using synthetic oligodeoxynucleotides (ODNs) are currently being evaluated in clinical trials for cancer, inflammation, and viral diseases. These macromolecules afford a unique opportunity to treat disease at the molecular level. The specificity of these compounds is derived from the genetic code and Watson-Crick base pairing, utilizing an antisense paradigm for the inhibition of translation and the regulation of protein expression. Currently, most antisense ODNs in development contain a phosphorothioate (P=S) backbone. Additional modifications primarily involve the 2' position on the ribose or modification of the nucleotide linkages of the backbone. To date, no toxicities in animal models appear related to inhibition of the pharmacologic target, rather toxicities induced by P=S ODNs appear similar and are independent of pharmacologic target. In general, toxicities correlate well with pharmacokinetic or tissue distribution parameters. In primates, the primary acute effects are associated with complement activation and the systemic effects associated with accumulation of high concentrations of P=S ODNs in the kidneys. In rodents, the primary effect is an immune stimulation characterized by splenomegaly, lymphoid hyperplasia, and mononuclear cell infiltrates in multiple tissues. At extraordinarily high doses (15-50 times the targeted clinical doses), hepatocellular and renal tubular degeneration are evident in rodents. Second generation antisense compounds, new routes of administration, and new formulations appear to broaden and improve the application of antisense technology.

Evaluation of the renal effects of an antisense phosphorothioate oligodeoxynucleotide in monkeys.

Antisense phosphorothioate oligodeoxynucleotides are therapeutic agents that provide target specificity resulting from Watson-Crick base pairing. However, there are nonspecific effects that in some instances result in toxicity. These compounds accumulate in the kidney and induce renal proximal tubular degeneration at high doses. The relationship between accumulation of phosphorothioate oligodeoxynucleotides in the kidney, indicators of renal toxicity, and histomorphology were investigated in rhesus monkeys. Monkeys received vehicle or an escalating dose regimen of 3, 10, 40, and 80 mg/kg of ISIS 2105 and were then evaluated for changes in clinical pathology indices, urinalysis parameters, and renal histopathology. Urinalysis revealed an increase in protein levels and a slight increase in blood content following the third 40 mg/kg dose and continuing through the 80 mg/kg doses, whereas other urinary markers of renal toxicity were unchanged. Creatinine clearance was slightly decreased in monkeys during the 80 mg/kg dose cycle. Granulation in the cytoplasm of proximal tubular epithelial cells was evident by microscopic examination of kidney and was present at all doses examined and increased with dose. Immunohistochemical staining localized the oligodeoxynucleotide within these granules. Histopathologic changes consisting of minimal to moderate tubular degeneration were present only at the higher doses of 40 and 80 mg/kg and at high tissue concentrations, and these changes occurred concurrent with functional alterations, whereas lower doses (< or = 10 mg/kg) did not affect a pathologic or functional change.

In vivo distribution and metabolism of a phosphorothioate oligonucleotide within rat liver after intravenous administration.

In the rat, the liver represents a major site of phosphorothioate oligodeoxynucleotide deposition after i.v. administration. For this reason, we examined the intracellular fate of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, isolated from parenchymal and nonparenchymal cell types after systemic dosing using established perfusion and separation techniques followed by CGE. Isolated cells were further fractionated into nuclear, cytosolic and membrane constituents to assess the intracellular localization, distribution and metabolic profiles as a function of time and dose. After a 10-mg/kg i.v. bolus, intracellular drug levels where maximal after 8 hr and diminished significantly thereafter, suggesting an active efflux mechanism or metabolism. Nonparenchymal (i.e., Kupffer and endothelial) cells contained approximately 80% of the total organ cellular dose, and this was equivalently distributed between the two cell types, while the remaining 20% was associated with hepatocytes. Nonparenchymal cells contained abundant nuclear, cytosolic and membrane drug levels over a wide dose range. In contrast, at doses of less than 25 mg/kg, hepatocytes contained significantly less drug with no detectable nuclear-association. Doses at or above 25 mg/kg appeared to saturate nonparenchymal cell types, whereas hepatocytes continued to accumulate drug in all cellular compartments, including the nucleus. Our results suggest that although pharmacokinetic parameters vary as a function of hepatic cell type, significant intracellular delivery can be readily achieved in the liver after systemic administration.

Functional and subcellular organelle changes in isolated rat and human hepatocytes induced by tetrahydroaminoacridine.

Tacrine (tetrahydroaminoacridine) is a reversible cholinesterase inhibitor used for the treatment of Alzheimer's disease. This drug causes an elevation of serum aminotransferases in a limited population of patients. Several in vivo studies failed to elucidate the mechanism for the enzyme elevation but previous in vitro studies have indicated defects in mitochondrial function. In this study, electron microscopic, histochemical, and confocal microscopy techniques were used with primary hepatocyte cultures from humans and rats to examine the sequence of early cellular changes after tacrine exposure. Changes included ribosome alterations as early as 1-2 h following tacrine exposure at concentrations ranging between 0.1 and 1.0 mM. Mitochondrial membrane potential was also altered as indicated by decreased rhodamine 123 uptake with time. Cellular lysosome content increased as indicated by increased staining of fluorescein isothiocyanate (FITC)-conjugated dextran. The results of acid phosphatase histochemistry correlated with the FITC-dextran findings. Additionally, tacrine-related degranulation and vesiculation of the endoplasmic reticulum paralleled the ribosomal and mitochondrial changes. These subcellular changes were reproducible in rat and human hepatocytes, showing for the first time that human hepatocytes can be altered by tacrine. The molecular mechanism of the organelle changes is unknown at this time. Also, the relationship between these subcellular changes in isolated hepatocytes and the transaminase elevation noted in human populations treated with tacrine needs to be clarified.

Preclinical evaluation of the effects of a novel antisense compound targeting C-raf kinase in mice and monkeys.

CGP 69846A (ISIS 5132) is an antisense phosphorothioate oligodeoxynucleotide which targets human C-raf kinase and is currently being developed as an antineoplastic agent. The toxicity of this compound was evaluated in mice and monkeys following repeated i.v. injections or infusions for 4 weeks at doses up to 100 mg/kg. Because CGP 69846A is inactive in the mouse, ISIS 11061, the murine-specific homologue targeting C-raf kinase mRNA was evaluated concurrently with CGP 69846A to assess the potential toxicity associated with reduced C-raf expression. There were no toxicities that differentiated ISIS 11061 from CGP 69846A in mice. Effects in mice included hepatomegaly and hepatocellular degeneration at the high dose of 100 mg/kg CGP 69846A that potentially resulted in lethality. Other effects which were observed at 20 and 100 mg/kg included mononuclear cell infiltrates in multiple organs, extramedullary hematopoiesis in the spleen and liver, an increase in bone marrow cellularity, an increase in white blood cells, a decrease in platelet counts, and Kupffer cell hyperplasia. These alterations were reversible following a recovery period. No adverse effects in mice were observed with doses < or = 10 mg/kg. In monkeys, administration of 10 mg/kg of CGP 69846A was associated with effects observed with other P = S ODNs, namely, prolongation of activated partial thromboplastin time (APTT) and activation of complement. These effects were transient and correlated with plasma concentrations of CGP 69846A. Below a concentration of 35 micrograms/ml of intact CGP 69846A the prolongation of APTT was less than 50% and levels of complement split products were not increased. All monkeys tolerated complement activation with no evidence of treatment-related clinical signs. Complement and coagulation were not affected by the lower doses of 1 and 3 mg/kg. No histopathology or alteration in hematology or serum chemistry was induced by doses up to 10 mg/kg in monkeys. The plasma and tissue deposition of CGP 69846A were characterized in mice and monkeys and toxicity was dependent on dose of CGP 69846A. In the present preclinical evaluation of toxicity in mice and monkeys, CGP 69846A is well tolerated at doses targeted for clinical trials. Toxicities induced by CGP 69846A in monkeys and mice occurred at doses of 10 mg/kg and greater. Effects induced by CGP 69846A were not unique and have been observed previously with other phosphorothioate oligodeoxynucleotides.

Immune stimulation--a class effect of phosphorothioate oligodeoxynucleotides in rodents.

Treatment of rodents with phosphorothioate oligodeoxynucleotides induces a form of immune stimulation characterized by splenomegaly, lymphoid hyperplasia, hypergammaglobulinemia and mixed mononuclear cellular infiltrates in numerous tissues. Immune stimulation was evaluated in mice with in vivo and in vitro studies using a review of historical data and specific in vivo and in vitro studies. All phosphorothioate oligodeoxynucleotides evaluated induced splenomegaly and B-lymphocyte proliferation. Splenomegaly and B-lymphocyte proliferation increased with dose or concentration of oligodeoxynucleotide. Splenomegaly appeared to occur, at least in part, as a result of stimulation of B-lymphocyte proliferation. There were differences with respect to degree or potency of immune stimulation by different oligodeoxynucleotides. The rank order potencies for B-lymphocyte proliferation in vitro and splenomegaly correlated well for the oligodeoxynucleotides tested. Particular oligodeoxynucleotide sequence motifs or palindromes have been demonstrated to affect in vitro cell proliferation. Inclusion of a 5'-AACGTT-3' palindrome in a phosphorothioate oligodeoxynucleotide sequence significantly enhanced the potency. While inclusion of this palindrome or a CpG motif alone may contribute to the immune stimulation, these palindromes and motifs were clearly not the sole factor required for immune stimulation. Several phosphorothioate oligodeoxynucleotides that did not contain a CpG motif still induced immune stimulation in mice. The immune stimulation induced by phosphorothioate oligodeoxynucleotides was an effect of this class of compounds to which rodents are acutely sensitive.

Comparison of tacrine-induced cytotoxicity in primary cultures of rat, mouse, monkey, dog, rabbit, and human hepatocytes.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, an indication of potential hepatotoxicity. The mechanism of human hepatoxicity has been difficult to study, because of the absence of an animal model. Therefore, this study compared the cytotoxicity induced by tacrine in primary rat, mouse, monkey, dog, rabbit and human hepatocytes to determine differences in response to tacrine between species in vitro. Cytotoxicity was assessed by determination of extra- and intracellular lactate dehydrogenase. The ratio of intracellular enzyme to total enzyme (i.e. intracellular and extracellular) was used to represent the viabilities of the cultures. Concentration-dependent cytotoxicity occurred after four and 24-hour exposure over a tacrine concentration range of 0 to 380 micrograms/ml. Cytotoxic potency of tacrine in hepatocytes from human, dog, mouse and rat was not significantly different; monkey hepatocytes appeared slightly more sensitive, while rabbit hepatocytes appeared slightly less sensitive than human hepatocytes. Increased time of exposure to tacrine decreased the concentration necessary to induce a cytotoxic response. This in vitro model suggests only minimal differences in sensitivity to tacrine-induce cytotoxicity; therefore, cytotoxicity in primary cultures of hepatocytes from various species would appear to be related to common metabolite(s) and/or mechanism of cellular injury.

Cytotoxicity study of tacrine, structurally and pharmacologically related compounds using rat hepatocytes.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, as an indication of potential hepatotoxicity. However, acute and chronic studies with a limited number of animal models have not demonstrated hepatotoxicity. The present study compared the cytotoxicity in hepatocyte cultures of tacrine with structurally (proflavine and 9-aminoacridine) or pharmacologically similar compounds (physostigmine), as well as structurally modified tacrine to determine if there was a structure activity relationship with regards to toxicity. Cytotoxicity was assessed by determination of extra- and intracellular amounts of lactate dehydrogenase. Cytotoxicity was assessed after a four-hour exposure over a test compound concentration range of 0 to 3 mM. Concentration-dependent cytotoxicity occurred with tacrine and all structurally related compounds. Physostigmine which is pharmacologically similar, but structurally different, did not induce cytotoxicity. Cytotoxic potency did not appear to be related to acetylcholinesterase inhibitory activity, while compounds with acridine structures induced cytotoxicity. Thus, in this in vitro model, cytotoxicity appears to be related to structure and not pharmacological action. Results of this study indicate that compounds structurally related to tacrine are cytotoxic because of the heterocyclic ring structure. Neither unsaturation of an aromatic ring of the heterocyclic compound, amino substitution of the heterocyclic rings, N-hydroxylation of the amino group, nor ring hydroxylation dramatically alter cytotoxicity.

Comparison of the microgel electrophoresis assay and other assays for genotoxicity in the detection of DNA damage.

Genotoxic agents can be detected by measuring DNA damage which result in the migration of DNA from single cells in agarose, using an electrophoretic field under alkaline conditions. The alkaline microgel electrophoresis technique was compared with in vitro structural chromosomal aberration (SCA) and mutation assays using V79 Chinese hamster lung cells and in vivo assays such as the bone marrow micronucleus assay in mice and a hepatocyte DNA repair assay in rats. Genotoxicants tested were those routinely used as positive control compounds in the various assays. In vitro assays included liver S9 for metabolic activation of cyclophosphamide (CP) for the SCA assay and benzo[a]pyrene (BP) for the mutation assay. A highly significant increase in DNA migration was induced by these agents under circumstances where a significant increase in DNA damage was detected using other endpoints. The alkaline microgel electrophoresis assay thus demonstrated the ability to detect DNA damage coinciding with the induction of DNA damage detected in these other assays for genotoxicity.

Degradation and inactivation of antitumor drugs.

Chemical methods for the degradation of 11 antineoplastic drugs [etoposide, teniposide, bleomycin, mitomycin C, cisplatin, cis-dichloro-trans-dihydroxy-bis(isopropylamine) platinum IV (CHIP), cyclophosphamide, ifosfamide, carmustine, lomustine, and methotrexate] were investigated. The success of the degradation procedures was assessed by HPLC and degree of biological inactivation by mutagenicity assays. The most widely applicable procedure was oxidation with potassium permanganate or 5.25% sodium hypochlorite solution (bleach). Oxidation completely degraded and inactivated etoposide, teniposide, bleomycin, mitomycin C, and methotrexate. In addition, oxidation followed by nucleophilic substitution resulted in the complete degradation and inactivation of cyclophosphamide and ifosfamide. Although carmustine and lomustine were chemically degraded by treatment with acidic potassium permanganate, the resulting reaction mixtures remained mutagenic. Therefore, this procedure cannot be recommended. The platinum-containing compounds, cisplatin and CHIP, were rendered nonmutagenic by reaction with sodium diethyldithiocarbamate. These easily performed, relatively safe procedures can be used to prevent exposure to mutagenic wastes and spills in the hospital setting.

Inhibition of sulfotransferase affecting unscheduled DNA synthesis induced by 2-acetylaminofluorene: an in vivo and in vitro comparison.

The unscheduled DNA repair (UDS) assay was conducted using the in vivo and in vitro procedures to investigate the role of arylsulfotransferases (AST) in the genotoxicity of 2-acetylaminofluorene (AAF). The in vivo assay had 4 groups of rats that consisted of those treated with pentachlorophenol (PCP), PCP and AAF, or AAF and an untreated control. The in vitro assay used hepatocytes from 3-methylcholanthrene or corn oil (control) treated rats. In both the in vivo and in vitro UDS assays AAF induced DNA damage. PCP, an inhibitor of arylsulfotransferase, significantly decreased AAF induced DNA damage. In the in vivo assay, PCP induced a significant increase in UDS and confounded an investigation of the role of sulfotransferase. The in vitro UDS assay more clearly defined the effect of PCP on AAF genotoxicity.

Inhibition of sulfotransferase in primary cultures of human hepatocytes affecting metabolism and binding of 2-acetylaminofluorene.

The metabolism and DNA binding of acetylaminofluorene (AAF) was investigated in human hepatocytes that were isolated from donor liver tissue by collagenase perfusion. Hepatocytes were treated with 0.01 microM pentachlorophenol (PCP), as a sulfotransferase inhibitor, to investigate the role of sulfotransferase in human bioactivation of aromatic amines. Concentrations of PCP greater than 0.1 microM resulted in cytotoxicity as noted by detachment of cells and atypical morphology. The metabolites of AAF were identified by HPLC as aminofluorene, 7-OH-AAF, 9-OH-AAF, 5-OH-AAF, N-OH-AAF, 1-OH-AAF and 3-OH-AAF. No consistent alteration in the metabolites produced occurred with PCP treatment compared to controls. PCP treatment increased total DNA binding of AAF metabolites compared with controls, suggesting that sulfotransferase does not activate AAF in human hepatocytes. Inhibition of sulfotransferase in human hepatocytes does not decrease DNA binding of AAF metabolites as noted previously with rat hepatocytes. Therefore, PCP may inhibit a detoxication pathway. This study supports N,O-acyltransferase as the critical enzyme for the formation of the major reactive metabolite in human liver.

Carcinogen-DNA adducts in cultures of rat and human hepatocytes.

Exposure to chemical carcinogens can often be identified by detection of DNA adduct lesions. Primary cultures of isolated rat and human hepatocytes were exposed to 2-acetyl-aminofluorene (AAF), 4-aminobiphenyl (ABP), or benzo[a]pyrene (BP). The isolated DNA from the exposed cells was analyzed using the 32P-post-labeling assay. A greater total of carcinogen-DNA adducts, 2-12-fold, were observed in human hepatocytes than rat hepatocytes at the same concentrations. The predominant DNA adducts for each carcinogen were the same between rat and human cells. The N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major AAF-DNA adduct. The N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the major ABP-DNA adduct. In the rat N2-[10 beta-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl] deoxyguanosine (dG-N2-BP) and two unidentified adducts were nearly equivalent in amount, while the major BP-DNA adducts in the humans was the dG-N2-BP. The rat hepatocyte in vitro results are comparable to the predominant adducts found with rats exposed in vivo. The two different cultures of human hepatocytes demonstrated qualitative and quantitative differences in specific DNA adducts from rat hepatocytes. This study and others using human hepatocyte cultures demonstrate that this in vitro system can provide useful information for assessing human carcinogenic hazards.

Genotoxicity assessment of pirmenol, a new antiarrhythmic drug.

The genotoxicity of pirmenol was tested in the E. coli and S. typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay. The E. coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Five strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9. Pirmenol was not mutagenic toward either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+). Pirmenol increased the frequency of structural chromosome aberrations (SCAs). The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations. Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory. The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks. Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity. This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing. Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay.

Induction of cytochrome P(1)450 RNA and benzo[a]pyrene metabolism in primary human hepatocyte cultures with benzanthracene.

Exposure of cells to microsomal enzyme inducers can modify the potency of many carcinogens. We have examined the steady-state level of RNA from the P(1)450 gene and the metabolism of benzo[a]pyrene (BP) in primary cultures of human hepatocytes exposed for up to 4 days to 12.5 microM benzanthracene (BA), and in uninduced control cultures. While the steady-state levels of RNA from the P(1)450 gene were nondetectable in uninduced (DMSO only) human hepatocytes, 12.5 microM BA-induced AHH activity, BP metabolism, and/or P(1)450-specific RNA in hepatocytes from seven human cases were investigated. RNA levels specific for the P(1)450 gene appeared maximal at 24 hr following exposure to BA, whereas, the protein, as determined by AHH enzyme activity from BA-induced hepatocytes, continued to increase up to the last time point examined, 72 hr. BA induction for 96 hr increased metabolism of BP (initial concentration of BP, 10 microM) over a time course of 3, 6, 12, and 24 hr of incubation with BP compared with that of controls. The major metabolites of BP produced by human hepatocytes in culture were the unidentified polar BP metabolite(s), possibly polyhydroxylated. BA induction caused approximately a twofold increase in these metabolites. BA-induced cultures showed an increase in glutathione conjugation compared to that in controls. The percentage of glucuronide and sulfate conjugates remains similar in all cultures. Total binding of tritium label BP to DNA was 1.3-fold to fivefold greater in induced cultures, and related more to total metabolism than to production of a specific metabolite. Exposure of human hepatocytes in vitro to BA leads to a large increase in the steady-state level of the RNA specific for the P(1)450 gene and an increase metabolism of BP.

Conjugation of chemical carcinogens by primary cultures of human hepatocytes.

1. The conjugation of benzo[a]pyrene (BP) and 2-acetylaminofluorene (AAF) was investigated in primary cultures of human hepatocytes. Human hepatocytes conjugated 12.5-63% of the BP and 1.7-52% of the AAF to sulphates and glucuronides over a thousand-fold concentration range. 2. BP is conjugated to glucuronides from non-detectable levels to 50%, and to sulphates from non-detectable levels to 30%. The major conjugated metabolites are the highly polar metabolites. 3. AAF is conjugated to glucuronides from 1.5 to 51% and to sulphates from 0.2 to 12%. The C-hydroxylated AAF metabolites were conjugated to glucuronides more than N-hydroxy AAF and aminofluorene metabolites.

A comparison of the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and DNA-binding of 2-acetylaminofluorene.

The metabolism of 2-acetylaminofluorene (AAF) in primary cultures of rat and human hepatocytes was investigated to determine if the activation of this well-studied chemical carcinogen proceeds via similar routes of metabolism between species. The total level of AAF metabolite(s) bound to hepatocellular DNA was determined in the presence of deacetylase inhibitors, diethyl(p-nitrophenyl) phosphate (paraoxon) or bis(p-nitrophenyl) phosphate (BPNPP). These compounds are known to inhibit deacetylase and to decrease the mutagenicity of AAF. Experiments with rat and human hepatocytes demonstrated inhibition in the deacetylation of AAF (5 x 10(-4) M) with paraoxon or BPNPP. The BPNPP (5 x 10(-4) M inhibited 99% of the AF formation in the human hepatocytes and 88% inhibition in the rat hepatocytes. Paraoxon at 10(-4) M demonstrated a 98% inhibition of deacetylation with humans and a 92% inhibition with rats. The rat hepatocytes also showed a 53% decrease in DNA binding in the presence of paraoxon. In contrast with human hepatocytes, while paraoxon decreased the AF metabolite by greater than 97%, there was no change in total DNA binding.