Dose response of hepatocyte replication in rats following continuous exposure to diethylnitrosamine.

Chronic exposure to the hepatocellular carcinogen diethylnitrosamine (DEN) causes a dose-dependent accumulation of the promutagenic DNA adduct O4-ethyldeoxythymidine in hepatocytes and increases in the number of initiated hepatocytes as indicated by gamma-glutamyl transpeptidase positive foci. Initiation is thought to be dependent on the quantity of promutagenic DNA adducts, their efficiency for causing base pair mismatch, and the extent of replication in the target tissue. If the extent of replication is also dose dependent, then this dependence could alter the number of promutagenic DNA adducts that mispair prior to repair and enhance the clonal expansion of initiated cells. We have examined the effect of DEN on hepatocellular proliferation over a wide range of doses. Six-week-old male F-344 rats were exposed to drinking water containing 0.4, 1, 4, 10, 40, or 100 ppm DEN for 1, 4, or 10 weeks. Following exposures to DEN, rats were injected i.p. with [3H]thymidine, sections from the left, right median and anterior right lobes of the liver were processed for autoradiography and the labeling index of hepatocytes determined. A progressive increase in hepatocyte replication was induced by exposure to 40 and 100 ppm DEN. This was especially marked in the left lobe where 40 and 100 ppm DEN induced increases of 800 and 1500%, respectively, over controls after 10 weeks of exposure. Exposure to 4 and 10 ppm DEN resulted in a 300 to 400% increase in hepatocyte replication in all lobes, whereas 1 and 0.4 ppm DEN did not significantly increase cell proliferation compared to unexposed controls.

Molecular dosimetry of O4-ethyldeoxythymidine in rats continuously exposed to diethylnitrosamine.

There is considerable interest in incorporating mechanistically based biological data into the process of quantitative risk assessment. Presently, no adequate data bases for internal dosimeters, such as DNA adducts, exist for humans or experimental animals. Therefore, the major promutagenic ethyl adduct, O4-ethyldeoxythymidine (O4-EtdT), has been quantitated in liver DNA after continuous exposure of rats to drinking water containing 0.4, 1, 4, 10, 40, or 100 ppm diethylnitrosamine (DEN) for 1, 4, 7, 14, 28, 49, or 70 days. The rate of O4-EtdT accumulation was modeled as the difference between a DEN-dependent rate of formation and an O4-EtdT concentration-dependent rate of loss. In general, O4-EtdT concentrations increased rapidly during the first 7 days of exposure and by 7-28 days O4-EtdT had accumulated to apparent steady-state concentrations that were DEN concentration-dependent over the entire dose range. The concentration of the adduct increased with DEN concentration over the entire dose range for exposures of 28 days or less and for doses of 0.4 to 40 ppm DEN the adduct level increased with DEN concentration for exposures of 70 days or less. Although the dose response of O4-EtdT was relatively linear, with increasing DEN concentration a trend toward a less than linear relationship was observed. This suggests that there was a lower efficiency of formation and/or greater loss of O4-EtdT during high-dose exposures. This study provides a data base that can be used to qualitatively and quantitatively examine the relationship between external dose and O4-EtdT over a DEN dose range covering several orders of magnitude.

Accumulation and persistence of DNA adducts in respiratory tissue of rats following multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.

4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major nitrosamine formed in tobacco smoke, induces a high incidence of lung, liver, and nasal cavity tumors in rats. Since alpha-hydroxylation of NNK by target tissues can lead to the generation of a methylating agent, the formation and removal of 7-methylguanine and the promutagenic lesions O6-methylguanine (O6mGua) and O4-methyldeoxythymidine were determined over 12 days of NNK administration to rats (100 mg/kg/day). DNA alkylation was greatest in the nasal mucosa, followed by liver and lung after 1 dose of NNK. No DNA adducts were detected in kidney and brain under these conditions. The concentration of O6mGua increased steadily in lung throughout the treatment regimen, while O6-methylguanine-DNA methyltransferase decreased to less than 5% of control. The concentration of O4-methyldeoxythymidine in lung DNA reached a steady state after 4 days of carcinogen treatment. After NNK treatment was discontinued, O6mGua persisted, while O4-methyldeoxythymidine was removed rapidly in the lung, suggesting that different repair pathways exist for the removal of these adducts in vivo. In hepatocytes, nonparenchymal cells, and nasal mucosa, O6mGua concentrations were maximal after 1-2 days and declined by 50-80% during the remaining 10 days of treatment. The decrease in O6mGua levels in nasal mucosa paralleled a decline in O6-methylguanine-DNA methyltransferase activity and was associated with marked cytotoxicity to Bowman's glands, portions of the lateral nasal gland, and the olfactory and respiratory mucosa during carcinogen treatment. In contrast, the decline in O6mGua in hepatocytes was attributed to the induction of O6-methylguanine-DNA methyltransferase activity, since an 18-fold reduction in the ratio of O6mGua:7-methylguanine was observed over the 12 days of treatment. These studies have demonstrated a marked accumulation of promutagenic DNA adducts in target tissues during repeated exposure to NNK.

Hepatocyte initiation during continuous administration of diethylnitrosamine and 1,2-sym-dimethylhydrazine.

Hepatocyte initiation, as indexed by growth-selected gamma-glutamyl transferase-positive foci, was measured during continuous exposure to diethylnitrosamine (DEN) at concentrations used in previous DEN bioassay, DNA adduct, and cell replication studies. Hepatocyte initiation increased in a dose- and lobe-specific manner. The efficiency of DEN as an initiating agent was not affected by DEN dose rate over concentrations ranging from 4 to 40 ppm. In a similar experiment the initiating abilities of 1,2-sym-dimethylhydrazine and DEN were compared under continuous exposure regimens. Foci induction increased in a lobe- and time-dependent manner. When left and median lobes were compared, the initiating ability of the two compounds correlated with their carcinogenicity. However, when corresponding anterior lobes were compared, no such correlation was observed.

Effect of a template-located 2',2'-difluorodeoxycytidine on the kinetics and fidelity of base insertion by Klenow (3'-->5'exonuclease-) fragment.

Gemcitabine [2',2'-difluorodeoxycytidine (dFdCyd)], a potent antitumor agent, inhibits DNA synthesis and is incorporated internally into DNA. The effect of a template-incorporated dFdCyd molecule (dFdCyd-) on DNA polymerase function was examined. Two 25-base deoxyoligonucleotides were synthesized with either a single dFdCyd- or template-incorporated deoxycytidine molecule (dCyd-) at the same position. Each was annealed separately to an identical complementary 5'-32P-labeled primer and extended by the Klenow fragment (3'-->5' exo-) of DNA polymerase I. "Correct" insertion of dGMP was 80-fold less efficient opposite dFdCyd- than dCyd-. A comparison of misinsertion efficiencies opposite template dFdCyd gave values of 2.7 x 10(-2) for dAMP insertion, 1.1 x 10(-3) for dTMP insertion, and 5.9 x 10(-4) for dCMP insertion. A similar measurement opposite template dC gave values of 1.8 x 10(-4), 1.7 x 10(-4), and 2.9 x 10(-6) for dAMP, dTMP, and dCMP insertion, respectively. Thus, the presence of dFdCyd on the template strand inhibited "normal" DNA synthesis and increased deoxyribonucleotide misinsertion frequencies. Pausing during DNA synthesis occurred directly opposite template dFdCyd suggesting that dFdC.dG base pairs might be less stable than normal dC.dG pairs, resulting in a decreased rate of primer extension beyond this site. Consistent with kinetic data, thermal denaturation measurements using comparable surrounding sequences showed that dFdC.dG "correct" pairs were less stable than dC.dG base pairs. Measurements on base mispairs showed that dFdC.dC was more stable than dC.dC, while no measurable Tm differences were found between polymers containing dFdC.dA and dC.dA or dFdC.dT, and dC.dT.

Antitumor efficacy, pharmacokinetics, and biodistribution of NX 211: a low-clearance liposomal formulation of lurtotecan.

Lurtotecan is a clinically active water-soluble camptothecin analogue that has been formulated into a low-clearance unilamellar liposome, NX 211. Comparative studies between free drug and NX 211 have been performed assessing pharmacokinetics in nude mice, tissue distribution in tumor-bearing mice, and antitumor efficacy in xenografts. Compared with lurtotecan, NX 211 demonstrated a significant increase in plasma residence time and a subsequent 1500-fold increase in the plasma area under the drug concentration curve. The volume of distribution was also greatly restricted, suggesting altered tissue distribution. Evaluation of tissues 24 h after administration of either [14C]NX 211 or [14C]lurtotecan to ES-2 tumor-bearing mice demonstrated a 40-fold increase in radiolabeled compound in the tumors of NX 211-treated mice compared with mice treated with lurtotecan. In single-dose efficacy studies, NX 211 produced a consistent 3-fold or greater increase in therapeutic index compared with lurtotecan in both the KB and ES-2 xenograft models. When compared at equitoxic levels in repeat-dose efficacy studies, NX 211 generated durable cures lasting >60 days and a 2-8-fold increase in log10 cell kill, compared with lurtotecan and topotecan, respectively. Together, these data demonstrate that NX 211 has significant therapeutic advantage over lurtotecan and that the improved antitumor activity is consistent with increased exposure and enhanced drug delivery to tumor sites.

Expression of TRPM-2 during involution and regeneration of the rat liver.

Increased message levels of testosterone-repressed prostate message-2 (TRPM-2) have been associated with programmed cell death in many tissues. To study its involvement in the apoptotic elimination of hepatocytes during liver involution and regeneration, levels of TRPM-2 message were evaluated in situ and by the ribonuclease protection assay. Although significant increases in apoptotic bodies were observed in rats 96 h following treatment with lead nitrate and ethylene dibromide, an increase in TRPM-2 message was not detected. Therefore, the expression of TRPM-2 mRNA may be a poor indicator of the extent to which apoptosis occurs during liver involution.

Polymerization of 2'-fluoro- and 2'-O-methyl-dNTPs by human DNA polymerase alpha, polymerase gamma, and primase.

Studies were undertaken to assess the ability of human polymerase alpha (pol alpha) and polymerase gamma (pol gamma) to incorporate 2'-fluoro- and 2'-O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine K(m) and V(max) for 2'-FdATP, 2'-FdUTP, 2'-FdCTP, 2'-FdGTP, 2'-O-MedATP, 2'-O-MedCTP, 2'-O-MedGTP, 2'-O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha incorporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma incorporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine substitution decreased V(max)/K(m) 2'-FdUTP. Because kinetics of insertion of pol alpha can be affected by the nature of the primer, we examined the ability of pol alpha to polymerize 2'-fluoro- and 2'-O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2'-FdATP and 2'-FdGTP were polymerized, but 2'-O-MedATP and 2'-O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2'-deoxy-2'-fluorocytosine (2'-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2'-FdNTPs but cannot polymerize 2'-O-MedNTPs.

Relationships between DNA adduct formation and carcinogenesis.

An impressive array of evidence has been obtained during the past decade establishing correlations between specific DNA adducts and carcinogenesis. Many of the studies utilized organ specific differences in carcinogenesis to establish the correlations. More recently, we have investigated similar relationships between target and nontarget cell populations within the liver. Chronic exposure to methylating hepatocarcinogens predominantly induces hemangiosarcomas, whereas exposure to ethylating agents causes hepatocellular carcinomas. This cell specificity in carcinogenesis correlates well with the presence of promutagenic DNA adducts. In the case of methylating agents, the nonparenchymal cells accumulate O6-methylguanine whereas the hepatocytes do not. Exposure to ethylating agents leads to accumulation of O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine in hepatocytes. These differences reflect the ability of the two cell populations to repair O6-alkylguanine and the extent of purine and pyrimidine alkylation with methylating and ethylating agents. Hepatocytes of rats exposed to diethylnitrosamine for 28 days have four to five times more promutagenic DNA adducts (O6-alkyldeoxyguanosine and O4-alkyldeoxythymidine) than hepatocytes of rats exposed to nearly equimolar doses of dimethylhydrazine. Both O6-methylguanine and O4-methyldeoxythymidine are rapidly repaired by rat hepatocytes, while only O6-ethyldeoxyguanosine is rapidly repaired. Studies comparing the relationship between the induction of gamma-glutamyl transpeptidase-positive foci, hepatocellular carcinoma and promutagenic lesions such as O4-ethyldeoxythymidine will be useful in understanding associations between the molecular dosimetry of DNA adducts, initiation, and progression of hepatocarcinogenesis.

High- to low-dose extrapolation: critical determinants involved in the dose response of carcinogenic substances.

Recent investigations on mechanism of carcinogenesis have demonstrated important quantitative relationships between the induction of neoplasia, the molecular dose of promutagenic DNA adducts and their efficiency for causing base-pair mismatch, and the extent of cell proliferation in target organ. These factors are involved in the multistage process of carcinogenesis, including initiation, promotion, and progression. The molecular dose of DNA adducts can exhibit supralinear, linear, or sublinear relationships to external dose due to differences in absorption, biotransformation, and DNA repair at high versus low doses. In contrast, increased cell proliferation is a common phenomena that is associated with exposures to relatively high doses of toxic chemicals. As such, it enhances the carcinogenic response at high doses, but has little effect at low doses. Since data on cell proliferation can be obtained for any exposure scenario and molecular dosimetry studies are beginning to emerge on selected chemical carcinogens, methods are needed so that these critical factors can be utilized in extrapolation from high to low doses and across species. The use of such information may provide a scientific basis for quantitative risk assessment.

Fialuridine is phosphorylated and inhibits DNA synthesis in isolated rat hepatic mitochondria.

Fialuridine (FIAU) is a thymidine analog effective against hepatitis B virus. Toxicity associated with FIAU treatment included clinical signs consistent with mitochondrial dysfunction, including severe lactic acidosis. To understand further the mechanism of FIAU toxicity, we examined the effect of FIAU on DNA synthesis in mitochondria. Mitochondria isolated from livers of naive rats were treated in vitro with concentrations of FIAU or FIAU triphosphate (FIAU-TP) ranging from 0.1 to 200 microM. A 14 or 32% decrease in mitochondrial DNA synthesis compared to controls was observed when isolated mitochondria were treated with 25 microM FIAU or FIAU-TP, respectively. Since it is thought that nucleosides must be phosphorylated to inhibit DNA polymerase, studies were conducted to determine whether isolated rat mitochondria could phosphorylate FIAU. Results using lanthanum chloride precipitation and HPLC analysis showed that enzymes present in a mitochondrial lysate were capable of phosphorylating FIAU to form FIAU monophosphate.

Modification of delayed rectifier potassium currents by the Kv9.1 potassium channel subunit.

Within auditory pathways, the intrinsic electrical properties of neurons, and in particular their complement of potassium channels, play a key role in shaping the timing and pattern of action potentials produced by sound stimuli. The Kv9.1 gene encodes a potassium channel alpha subunit that is expressed in a variety of neurons, including those of the inferior colliculus. When cRNA encoding this subunit is injected into Xenopus oocytes, no functional channels are expressed. When, however, Kv9.1 is co-expressed with certain other alpha potassium channel subunits, it changes the characteristics of the currents produced by these functional channel proteins. We have found that Kv9.1 isolated from a rat brain cDNA library alters the kinetics and the voltage-dependence of activation and inactivation of Kv2.1, a channel subunit that generates slowly inactivating delayed rectifier potassium currents. The rate of activation of Kv2.1 is slowed by co-expression with Kv9.1. With Kv2.1 alone, the amplitude of evoked currents increases monotonically with increasing command potentials. In contrast, when Kv2.1 is co-expressed with Kv9.1, the amplitude of currents increases with increasing depolarization up to potentials of only approximately +60 mV, after which increasing depolarization results in a decrease in current amplitude. Currents produced by Kv2. 1 alone and by Kv2.1/Kv9.1 are both sensitive to the potassium channel blocker tetraethyl ammonium ions (TEA), but higher concentrations of TEA (20 mM) eliminate the biphasic voltage-dependence of the Kv2.1/Kv9.1 currents. Co-expression with Kv9.1 also produces an apparent negative shift in the voltage-dependence of inactivation and activation. Computer simulations of model neurons suggest that co-expression of Kv9.1 with Kv2.1 may have different effects in neurons depending on whether their firing pattern is limited by the inactivation of inward currents. In excitable cells in which the inward currents do not inactivate, co-expression with Kv9.1 could produce an inhibition of firing during sustained depolarization. In contrast, in model neurons with rapidly inactivating inward current, the change in the voltage-dependence of activation produced by Kv9.1 may allow the cells to follow high frequency stimulation more effectively.

Sequence-dependent formation of alkyl DNA adducts: a review of methods, results, and biological correlates.

Understanding the influence of the DNA sequence on chemical-DNA interactions may provide insight into the processes of chemical carcinogenesis and mutagenesis. This article provides a brief overview of studies and methods devoted to examining the distribution of DNA adducts produced by alkylating agents. Particular emphasis is placed on discussion of DNA adducts generated by simple alkylating agents and the role that their distribution may play in the generation of mutational hotspots.

The evaluation of methapyrilene for bacterial mutation with metabolic activation by Aroclor-induced, methapyrilene-induced and noninduced rat-liver S9.

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.1% methapyrilene in the diet for 10 weeks prior to sacrifice. S9 was prepared from the livers of the control, MP-treated and Aroclor-induced Fischer 344 rats. Each type of S9 was analyzed for mixed function oxidase activity, cytochrome P-450, and protein content. MP was then evaluated for mutagenicity in 6 strains of S. typhimurium (TA1535, TA1537, TA98, TA100, TA2638 and TA104) and one strain of E. coli (WP2uvrA-) using the standard plate-incorporation assay. MP was not mutagenic in any of the 7 bacterial strains when tested at concentrations < or = 10 mg/ml in the presence of each type of S9. However, in the absence of metabolic activation, an approximate 2-fold increase in revertants was noted with strain TA1535. The data from this study show that MP was not converted to a mutagenic metabolite by any of the three S9 types examined. However, the "weak" positive response with strain 1535 in the absence of metabolic activation indicates that further research is needed to elucidate the mechanism of action of this rat carcinogen.

Incorporation of fialuridine (FIAU) into mitochondrial DNA and effects of FIAU on the morphology of mitochondria in human hepatoblastoma cells.

Fialuridine (FIAU), a thymidine nucleoside analogue with anti-hepatitis B virus activity, showed clinical toxicity consistent with mitochondrial dysfunction. In vitro methods were used to understand further this toxicity. Using a sensitive and specific radioimmunoassay, FIAU was found to be present in nuclear DNA of human hepatoblastoma cells incubated for 6 days in 10 or 50 n M drug, at a level of 1 residue per 63 or 39 thymidines, respectively, and was present in mitochondrial DNA at a level of 1 residue per 2139 or 1696 thymidines, respectively. Human hepatoblastoma cells were incubated for 6 days in increasing concentrations of FIAU or, for comparative purposes, the nucleoside analogue dideoxycytidine (ddC), after which time the cells were examined by electron microscopy. At 10 mum and higher concentrations, both compounds induced morphological changes in the ultrastructure of mitochondria characterized by marked mitochondrial swelling, loss of internal cristae and dissolution of the internal matrix. These results, considered along with previously published studies, indicate that FIAU has deleterious effects in vitro on mitochondrial function and structure that occur relatively quickly but without an apparent decrease in the abundance of mitochondrial DNA.

Alterations in DNA-restriction enzyme interactions by O4-alkyldeoxythymidines.

O4-Alkyldeoxythymidines have been extensively studied for their ability to cause mutations and to induce cancer. Since these adducts can change DNA conformation, they may also have a more immediate effect of altering DNA-protein interactions. To address this issue, the effects of these adducts on restriction enzyme activity were examined. Oligodeoxyribonucleosides containing O4-ethyldeoxythymidine (O4-EtdT) or O4-methyldeoxythymidine (O4-MedT) at a unique site within the sequence 5'-GAATGGATCCTAATGAGATC-3' were constructed by automated DNA synthesis. This sequence contains the recognition site for various restriction enzymes. These oligomers were annealed to various complementary strands and digested with restriction enzymes: BamHI or BstI (GGATCC); Sau3A, NdeII, or MboI (GATC); DpnI (GmATC); and BstYI, MflI, or XhoII (PuGATCPy). Analysis of the digests demonstrated that the presence of either O4-EtdT or O4-MedT abolished the ability of XhoII, MboI, MflI, or NdeII to cut at the restriction site. DpnI failed to cut any of the oligomers. BamHI, Sau3A, BstI, and BstYI exhibited alterations in cutting specificity depending upon the oligomers used. These results demonstrated that O4-alkyldeoxythymidine adducts alter DNA-restriction enzyme interactions in a protein- and sequence-dependent manner. Because of the importance of natural methylation in genetic regulation, it is possible that aberrant methylation in the form of DNA adducts could also alter protein-DNA interactions in cells exposed to DNA-modifying agents.