Reduction in cis-diamminedichloroplatinum(II)-induced cytotoxicity, sister chromatid exchange, and DNA interstrand cross-links in 9L cells treated with the polyamine biosynthesis inhibitor (2R,5R)-6-heptyne-2,5-diamine.

The effects of depletion of intracellular levels of the polyamines putrescine and spermidine on cis-diamminedichloroplatinum(II)-induced cytotoxicity, sister chromatid exchanges, DNA interstrand cross-linking, and intracellular glutathione levels were studied in 9L rat brain tumor cells pretreated with the ornithine decarboxylase inhibitor (2R,5R)-6-heptyne-2,5-diamine (R,R-MAP). Pretreatment of 9L cells with R,R-MAP for 48 h decreased cis-diamminedichloroplatinum(II) cytotoxicity with an average dose reduction ratio of 0.55 at both the 5 and 10% survival levels; addition of putrescine partially prevented this effect. The number of sister chromatid exchanges and DNA interstrand cross-links was also reduced (31 and 38%, respectively). Within 24 h of treatment with R,R-MAP, intracellular glutathione levels began to increase relative to untreated control cells and were significantly elevated in R,R-MAP-treated cells 48-72 h after addition of drug. We discuss several mechanisms by which polyamine depletion could reduce cis-diamminedichloroplatinum(II) toxicity.

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or alpha-difluoromethylornithine.

We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and alpha-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 microM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 x 10(5)), medium (5 x 10(5)), or high (2 x 10(6)) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes.

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.

Disturbances in DNA precursor metabolism associated with exposure to an inhibitor of poly(ADP-ribose) synthetase.

3-Aminobenzamide (3AB) is widely used as an inhibitor of poly(ADP-ribose) synthetase to study the effect of protein ribosylation on various cellular processes, but the specificity of its inhibition has not been demonstrated. We found that 3AB has a wide range of effects on DNA precursor metabolism, as determined by high-performance liquid chromatographic separation of deoxynucleosides derived from enzymatic digestion of cellular DNA. 3AB (10-20 mM) significantly reduced cell growth in human lymphoblastoid cells. Furthermore, the incorporation of [3H]deoxycytidine into DNA was significantly enhanced relative to incorporation of [3H]deoxythymidine, [3H]deoxyguanosine, and [3H]deoxyadenosine. Incorporation of fragments of [3H]glucose into the pyrimidine fraction of DNA was significantly inhibited relative to incorporation into the purine fraction. At only 1 mM, 3AB had a major inhibitory effect on the incorporation of the methyl group from [3H]methionine into deoxyguanosine, deoxyadenosine, and deoxycytidine, with 50% inhibition into deoxyguanosine and deoxyadenosine and 90% inhibition into deoxycytidine. The specificity of 3AB inhibition to poly(ADP-ribose) synthetase is therefore doubtful in view of this variety of metabolic effects, involving pyrimidine synthesis and de novo synthesis via the one-carbon pool.

Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures.

Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of [3H]thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

Do inhibitor studies demonstrate a role for poly(ADP-ribose) in DNA repair?

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, has been commonly used in attempts to demonstrate a regulatory role for the polymer during a late stage of repair. When a range of inhibitor concentrations was used paradoxical results were obtained. Up to 1 mM, 3-aminobenzamide appeared to reduce DNA break frequencies in cells damaged by methyl methane sulfonate; at doses of 2 mM and above, it appeared to increase break frequencies. In the high concentration range, many nonspecific side effects and cellular toxicity predominate. Evidence used to assert a role for poly(ADP-ribose) synthesis during ligation has usually been derived from experiments using high concentrations of 3-aminobenzamide, but these may be attributed to toxic side effects. 3-Aminobenzamide stimulates a large increase in repair replication which does not result from increased excision of damaged sites or an increased patch length but may be attributable to other cellular effects such as endogenous nuclease attack on DNA. The cellular effects of 3-aminobenzamide are therefore complicated by nonspecific effects over a commonly used concentration range and evidence for a specific regulatory role of poly(ADP-ribose) in DNA repair is weak.

A methodology for the analysis of the preneoplastic antigen.

A highly sensitive assay for the epoxide hydrolase activity associated with the preneoplastic antigen (PNA) has been developed based on the synthesis of cis-stilbene oxide labeled with tritium at approximately 15 Ci/mmol. This assay allows the detection of elevated epoxide hydrolase activity in the serum of humans and rodents as well as in the culture medium bathing isolated hepatocytes. The integrity of the enzymatic assay was confirmed in rodents by precipitating the serum PNA activity using an antibody raised against the rat microsomal epoxide hydrolase. Methodology for the detection of PNA in serum will facilitate evaluation of this antigen as a marker for hepatic neoplasia in man and in experimental animals.

Inhibitors of poly(adenosine diphosphate-ribose) synthesis: effect on other metabolic processes.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

Body composition and adiposity in LH-lesioned and pair-fed obese Zucker rats.

It has been reported previously that lateral hypothalamic (LH) lesions alter body composition including reducing adipocyte cellularity in lean and obese Zucker rats. The present experiment was designed to determine whether these alterations in body composition and adipose cell number are secondary to the reduced energy consumption of LH-lesioned rats or to a direct effect of the hypothalamic lesion. Groups of lean and obese Zucker rats, sustaining lesions of the lateral hypothalamus at 10 wk of age, maintained body weight at 72% that of nonlesioned controls until killed at 32 wk. Pair-feeding nonlesioned rats to the intakes of lean and obese LH-lesioned rats produced a reduction in adipocyte number similar to that caused by lesions. However, neither the lean nor obese LH-lesioned rats displayed an increase in cell number when fed a palatable diet that markedly increased carcass lipids. This finding suggests that adipocyte number may be constrained in LH-lesioned rats. These and further observations that 1) lean control rats maintained a higher body weight than the LH-lesioned rats to which they were pair fed and 2) in obese rats, food restriction further reduced protein deposition and elevated plasma insulin relative to comparably fed LH-lesioned obese rats suggest that the LH syndrome is mimicked by simple food restriction.

Isoproterenol alters nonshivering thermogenesis in the Zucker obese rat (fafa).

To test nonshivering thermogenic (NST) capacity of lean and obese Zucker rats, 4 doses of isoproterenol, ranging in concentration from 0.25 to 6.0 micrograms/min . kg . 75, were intravenously infused into 16 to 18 week old male rats, and oxygen consumption was continuously monitored. Obese rats had a decreased NST response relative to lean littermates. This lowered thermogenic response of the obese rats cannot be attributed to a decreased mass of brown adipose tissue since both the cervical and interscapular depots from obese rats weighed significantly more than did those from lean rats.

Developmental study of adipose cellularity in lateral hypothalamic-lesioned Zucker obese rats.

Ten-week-old lean and obese Zucker rats were sham lesioned or received bilateral, electrolytic lesions of the lateral hypothalamus (LH). They were maintained on a wet mash diet until killing at 15 or 32 wk of age; control lean and obese rats were also killed at 6 and 10 wk. Body composition analyses were performed and adipocyte cellularity of epididymal, retroperitoneal, and subcutaneous depots were calculated. Changes in body composition of LH-lesioned rats, though similar in the two genotypes on an absolute basis, differed on a percentage basis due to the extreme adiposity of the obese rats. Retarded development of protein depots in both lesioned lean and obese rats was apparent at 15 but not 32 wk. Relative to genotypic controls, lesioned lean and obese rats had smaller adipose depots by 32 wk due to decreased adipocyte size in lean rats and reduced adipocyte number in the obese. This genotype-specific response was probably due to the chronic hyperplasia of adipocytes unique to the obese rats. This distinctive developmental pattern of the epididymal depot is discussed.

Effect of lateral hypothalamic lesions on regulation of body weight and adiposity in rats.

Obese and lean Zucker rats received bilateral electrolytic lesions of the lateral hypothalamus (LH) at 10 wk of age; control obese and lean rats were sham lesioned. After lesioning the body weights of both obese and lean animals were first reduced and then maintained until being killed (32 wk) at a stable percentage of the nonlesioned control levels (74.5 and 78.3%, respectively). Carcass analysis revealed that the adipose tissue mass was significantly lowered by LH lesions in both the obese and lean animals. Percent carcass fat of lesioned lean rats was less than that of controls (15.0 vs. 23.5%) due to the presence of slightly, but not significantly, smaller and fewer adipocytes. Though absolute levels of fat were likewise lowered in obese rats, their percent carcass fat remained at control levels (52.0 vs. 53.0%) due to equivalent decreases in other body compartments. In the lesioned rats the reduced levels of adipose tissue were associated with a significant reduction in adipocyte number; adipocyte size was unchanged. It is concluded that the lateral hypothalamus of obese Zucker rats participates in the regulation of body weight in the same manner as in lean rats. The differences noted in percent carcass fat between LH lesioned lean and obese Zucker rats are apparently related to the obese animal's known propensity to sequester energy in the form of lipid.

Iron appetite and latent learning in rats.

Two experiments are reported which show that rats are capable of forming an association between the presence of iron in a solution when it is not specifically needed and a subsequent state of iron deficiency. Specifically, rats were trained to lever press for water while thirsty. One group received ferrous ions in addition to the water. When these rats were subsequently rendered iron deficient, they lever pressed more under extinction conditions as a graded function of lower hemoglobin levels. Controls that either did not receive ferrous ions during training or received solutions other than ferrous solutions during training did not respond this way under extinction conditions. This is therefore a type of latent learning previously demonstrated only for sodium appetite.