The effect of nitroheterocyclic drugs on DNA: an in vitro model of cytotoxicity.

Electrolytic reduction of several nitroheterocyclic drugs was carried out at controlled potentials under anoxic (anaerobic) conditions in the presence of Esherichia coli DNA. The DNA was examined during the reduction process by viscometry, determination of T_m values, and thermal renaturation of DNA; its single strand content was determined using hydroxyapatite chromatography and its electrophoretic migration characteristics in acridine-impregnated agarose gels. All the drugs examined decreased the viscosity, thermal renaturation and T_m value, and increased the single strand content and migration distance of DNA. The results indicate that the drugs cause strand breakage of DNA as a primary mechanism of action, resulting in extensive loss of helix formation and a concomitant decrease in molecular weight. The method of drug reduction and the techniques to assess DNA damage may be used as a model to determine the relative cytotoxicities and the potential of such agents as antimicrobial and radiosensitizing drugs.     

Interactions of lead and cadmium on acetylcholine release at the frog neuromuscular junction

The interactive effects of Cd2+ and Pb2+ on evoked and spontaneous transmitter release were studied in the sciatic nerve-sartorius muscle preparation of the frog (Rana pipiens). Either Pb2+ or Cd2+ competitively inhibited the actions of Ca2+ in bringing about evoked release, as measured by the endplate potential (EPP) amplitude. Combinations of Pb2+ and Cd2+ were additive in their effects on the EPP. The rate of spontaneous transmitter release was measured as the miniature endplate potential (MEPP) frequency. In contrast to their effects on the EPP, exposure of preparations to combinations of Pb2+ and Cd2+ actually increased the MEPP frequency less than exposures of the preparations to Pb2+ alone. The degree of reduction in MEPP frequency produced by Cd2+ depended upon the relative ratio of Pb2+ and Cd2+ ions. These results suggest that Pb2+ and Cd2+ ions competed for a common presynaptic receptor site during evoked release. They also suggest that Pb2+ ions may enter the nerve terminal, possibly through the Ca2+ channel, and that this entry is inhibited by Cd2+.     

Inhibition of delta-aminolevulinic acid synthetase by thioacetamide and thioacetamide-S-oxide.

Thiocetamide and one of its metabolites, thioacetamide-S-oxide, were shown to inhibit δ-aminolevulinic acid (ALA) synthetase when administered in vivo to adult male mice. Thioacetamide and thioacetamide-S-oxide also inhibited the 3,5-dicarboethoxy-1,4-dihydrocollidine (DDC)-mediated induction of ALA synthetase when given either 1hr prior to or 3 hr after the administration of DDC. The results of these studies also indicate that thioacetamide-S-oxide is generally a more potent inhibitor of ALA synthetase than thioacetamide.     

The effect of in vivo hyperoxic exposure on the release of endogenous histamine from the rat isolated perfused lung

Female rats were exposed to either 1 atm air or 100% O2 for 12, 24, or 48 hr. The rats were killed, the lungs were removed, and an isolated perfused lung (IPL) system was prepared. The isolated lung preparation was perfused with a modified Krebs-Henseleit buffer in a recirculating system, and the effect of the O2 exposures on histamine release from the IPL was determined. The effect of these O2 exposures on malondialdehyde formation in the IPL also was examined. Maximal release of histamine occurred after 20 min of perfusion. A linear relationship was found between the maximal histamine concentration released into the perfusate and the length of time the rats were exposed to normobaric hyperoxia. Malondialdehyde in lung perfusate also increased in a linear manner with increasing O2 exposure time. Addition of the H1-receptor antagonist, d-chlorpheniramine, to the perfusate completely inhibited basal histamine release from the IPL of both air- and O2-exposed rats, while addition of the H2-receptor antagonist, metamide, potentiated the release process. There was no significant effect demonstrated when an equimolar concentration of atropine was added to the perfusate. Arterial plasma histamine from rats exposed to 100% O2 for 24 hr increased 40% when compared to air-exposed controls, while histamine release from the IPL increased 75%. In conclusion, exposure of rats to normobaric hyperoxia caused both histamine release and malondialdehyde formation. Histamine release probably occurred as a result of a free radical-induced peroxidation of the lipid membrane of histamine-containing mast cells. Release of histamine from the IPL may be an early biochemical marker of damage by O2.     

The effect of ionophore A23187 and 2,4-dinitrophenol on the structure and function of cultured liver cells

Adult rat hepatocytes maintained in primary culture have been used as a model system to study cellular injury dependent upon extracellular calcium. Incubation of hepatocytes with ionophore A23187 (1 to 5 μm) resulted in leakage of cytoplasmic enzymes, an increase in the number of cells stained with trypan blue, blebing of the plasma membrane, and changes in mitochondrial structure characterized by mitochondrial swelling. Moreover, a 60% decrease in cellular ATP was observed to precede changes in cellular permeability. These cytotoxic alterations induced by ionophore were dependent upon the presence of extracellular calcium. In contrast, 2,4-dinitrophenol depleted ATP much more extensively and induced extensive swelling of mitochondria at concentrations which failed to induce significant leakage of cytoplasmic enzymes. Cytotoxic changes induced by ionophore were potentiated by 2,4-dinitrophenol but not ethionine which has been shown to cause a reduction in cellular ATP levels. However, ethionine potentiated the cytotoxicity induced by 2,4-dinitrophenol.     

The interaction of reduced metronidazole with DNA

Electrolytic reduction of metronidazole was carried out at a controlled potential under anaerobic conditions in the presence of calf-thymus DNA. The DNA was subsequently examined during the reduction process by viscometry, hyperchromicity, melting and renaturation profiles and by hydrolysis using DNAase 1. The reduced drug decreased the viscosity and thermal hyperchromicity of DNA, inhibited its renaturation and caused a 50 per cent increase in the length of oligonucleotides produced from DNA as a consequence of DNAase action. The results indicate that reduced metronidazole causes a loss of DNA helix content, strand breakage, and a concomitant impairment of its function as an enzyme template which may be due to the presence of altered bases or a drug-base complex. These findings can be used to explain the action of the drug both as a cytocidal antimicrobial agent and as a hypoxic call tumour radiosensitizer.     

Stimulatory effect of bleomycin on the hyaluronic acid synthetase in cultured fibroblasts.

The mechanism of the stimulatory effect of bleomycin on the production of hexosamine-containing substances by cultured fibroblasts was studied by measuring changes in the activity of hyaluronic acid (HA) synthetase. HA synthetase activity was increased by 1 gmg bleomycin/ml, and its stimulatory effect could be detected 4 days after the start of bleomycin treatment and was enhanced by lenthening the time of treatment, i.e. by 96 per cent for an 8-day treatment. In cell-free systems, however, bleomycin showed no stimulatory effect on this enzyme activity. The enhancement of HA synthetase activity by bleomycin was inhibited by the addition of cycloheximide or actinomycin D. The synthesis of HA was much less influenced by cycloheximide than was that og glycoprotein and sulfated glycosaminoglycan. From our results it can be concluded that HA synthetase activity, induced when the old medium of stationary cultures was renewed with fresh medium containing 5% fetal bovine serum, was significantly enhanced by the addition of bleomycin. However, the detailed mechanism of enhancement of HA synthetase activity has not yet been elucidated.     

The effect of application frequency on epidermal carcinogenesis assays

Classic mouse skin-painting assays of such highly-complex mixtures as petroleums and synthetic fuels may lead to severe local cytotoxic effects that can alter tumorigenesis. This effect can be particularly disruptive in dose-response studies where a wide range of doses is employed. The experiments described here illustrate that frequency of exposure may be more important than the concentration of individual doses. C3Hf/Bd mice were exposed to 2 shale oils in experiments in which a constant weekly dose was either applied once or divided into 2 or 4 equal parts and applied either twice or 4 times each week. In experiments with either oil, a single weekly application was more effective in total tumor production and less cytotoxic than more frequent application of less-concentrated doses.     

Acute effect of cadmium on hepatic drug-metabolizing enzymes in the rat

Single ip injections of cadmium chloride at doses of 2.5 or 3.75 mg/kg (equivalent to 1.5 or 2.3 mg of Cd, respectively) into male Wistar rats of mean body weight of 226 ± 17 g produced significant inhibition (25–60%) of aniline hydroxylase and nitroreductase activity and also lowered the microsomal cytochrome P-450 content to 50% of the control value. These effects are in contrasto those on O-demethylase activity, which was not inhibited by either dose of cadmium chloride when enzyme activities were subsequently measured in a 0.05 m phosphate-buffered incubation system. However, the opposite conclusion was reached for the effect of these same doses of cadmium chloride when O-demethylase activity was assessed in comparable Tris-buffered systems. There appears to be a definite “buffer effect” in operation here. While the higher dose of this toxic metal salt significantly reduced the activities of the phenobarbital-induced hepatic microsomal drug-metabolizing enzymes and of cytochrome P-450, the lower dose was without significant effect on this induction, with the one exception of aniline hydroxylase when assessed in a Tris-buffer system.     

Fate of aclacinomycin-A and its metabolites effect on cell growth and macromolecular synthesis

The relationship between the structure and activity of aclacinomycin-A (ACM) metabolites was investigated in vitro in Friend leukaemia cells (FLC). The cytotoxic effect was related to the ease with which ACM and its metabolites accumulate in the nucleus. Cellular uptake and nuclear incorporation are influenced by the hexopyranoses linked to aklavinone (AKV) and by the two methyls linked to the l-rhodosamine amino groups. The effect of ACM and its metabolites on macromolecular synthesis depended on the drug concentrations and the exposure time. ACM was the most active in the inhibition of nucleic acid synthesis whereas it had no direct effect on protein synthesis even at high drug concentrations. When cells were treated for a short time with low drug concentrations (1 μM), RNA synthesis was inhibited to a greater extent than DNA synthesis. But when incubated for longer periods, inhibition of DNA synthesis increased further. RNA and DNA syntheses were both inhibited to about the same extent only when cells were exposed to the higher drug concentrations (10 μM). We conclude therefore that at low drug concentrations the effect on DNA synthesis is probably a consequence of RNA synthesis inhibition. The early DNA synthesis inhibition which occurs at higher drug concentrations may result from the direct action on the cellular genome.     

Inhibition of nucleoside phosphorylase cleavage of 5-fluoro-2'-deoxyuridine by 2,4- pyrimidinedione derivatives

Several 2,4-pyrimidinedione (uracil) derivatives were evaluated as inhibitors of the pyrimidine nucleoside phosphorylases that cleave 5-fluorn-2'-deoxyuridine (FUdR) to 5-fluorouracil. Pyrimidinediones substituted at either N-1 or C-5, or both, markedly inhibited the phosphorolysis of FUdR by the uridine-deoxyuridine phosphorylases of Ehrlich ascites and Novikoff hepatoma cells. The most potent inhibitors were 5-benzyluracil derivatives substituted with alkoxy groups on the meta-position of the benzyl moiety; the most active of these was 5-{[3-(phenylmethoxy)phenyl]methyl}uracil. The same derivatives, however, did not inhibit the phosphorolysis of FUdR by the thymidine phosphorylases of murine liver, human leukocytes and HeLa (S3) cells. 6-Anilino and 6-(1-naphthylmethylamino) derivatives of uracil, which have been shown by others to inhibit the cleavage of FUdR by the thymidine phosphorylase activity of Escherichia coli, did not inhibit any of the mammalian thymidine or uridinedeoxyuridine phosphorylase activities. By contrast, pyrimidinediones substituted with smaller, non-hydrophobic groups at either C-5 or C-6, or both, inhibited the cleavage of FUdR by both the mammalian thymidine and uridine-deoxyuridine phosphorylases. The most active of these, 6-aminothymine, was also the best inhibitor of thymidine phosphorylase. Our results demonstrate differences in the active sites of the various pyrimidine nucleoside phosphorylases, and should provide a basis for the design of more potent and specific inhibitors of the nucleoside phosphorylase(s) responsible for the cleavage of FUdR in man.     

Effect of methionine deprivation on L5178Y murine leukemia cells in culture interference with the antineoplastic effect of methotrexate

L5178Y cells in culture have a requirement for l-methionine which cannot be satisfied by supplying the components necessary for de novo biosynthesis [1]. Methionine deprivation produced a rapid and progressive loss of cell viability (30°_o by 6 hr: 90°_o by 24 hr). Cells which remained viable after being deprived of methionine could be rescued by l-methionine supplementation.L5178Y cells in culture were also highly sensitive to the folate antagonist, methotrexate. Exposure to a concentration of 10^?6 M for 6 hr resulted in a 95–97°_o loss of viability. However, if cells were deprived of methionine for 6 hr before exposure to methotrexate, the methotrexate effect was reduced. The cell-killing effect of melhotrexate was blocked by longer intervals of methionine deprivation. If the deprived cells were resupplied with the amino acid, the effect of methotrexate was still reduced for at least 12 hr following the methionine resupplementation.     

Cardiac effects of 1-alpha-acetylmethadol. V. Possible involvement of the autonomic nervous system

The cardiovascular responses to 1-α-acetylmethadol (LAAM) and its two major metabolites, 1-α-acetylnormethadol (N-LAAM) and 1-α-acetyldinormethadol (DN-LAAM), were examined in anesthetized dogs. LAAM, N-LAAM, and DN-LAAM caused significant decreases (p < 0.05) in mean arterial blood pressure (BP), heart rate (HR), and myocardial contractile force (CF). N-LAAM appeared to be approximately 10 times more potent than LAAM or DN-LAAM in producing these effects. The cardiovascular responses to LAAM were also studied in vagotomized (VAGOT), chemically sympathectomized (SYMX), and vagotomized + chemically sympathectomized (VAGOT + SYMX) dogs. VAGOT had little effect on the responses to LAAM, SYMX and VAGOT + SYMX caused a decrease in the magnitude of the bradycardia produced by LAAM relative to that observed in intact animals and also increased the threshold dose required to produce significant decreases in BP; however, statistically significant (p < 0.05) decreases in BP, HR, and CF were observed. The data suggest that part of the hypotension and bradycardia produced by LAAM may be the result of sympathetic nervous system depression, but there also appears to be a direct component to the cardiovascular effects of LAAM and congeners.     

Long-term effects of an organophosphate upon the human electroencephalogram.

The brain electrical activity of workers exposed to the organophosphate compound (OP), sarin, was compared to that of control subjects. Exposed workers had a history of one or more documented accidental exposures to toxic levels of sarin. However, no exposed subject had exposure within 1 year of his examination. The comparison included standard clinical electroencephalograms (EEGs), computer-derived EEG spectral analysis, and standard overnight sleep EEGs. It was not possible to diagnose subjects individually by expert visual inspection of their EEGs. However, statistically significant between-group differences for both the visually inspected and computer-derived data were reported by both univariate and multivariate statistical methods. Different EEG changes revealed by visual inspection and computer-derived spectral analysis appear to reflect the differing sensitivites of these two analytic techniques. Statistically significant group differences included increased beta activity, increased delta and theta slowing, decreased alpha activity, and increased amounts of rapid eye movement sleep in the exposed population. It is suggested that the above findings represent an unexpected persistence of known short-term OP actions. It is also suggested that these results, when taken along with the reported long-term behavioral effects of OP exposure, provide parallel evidence that OP exposure can produce long-term changes in brain function.     

Behavioral effects of trimethyltin in two strains of mice. I. Spontaneous motor activity

Adult male mice (C57BL/6N and BALB/c) were administered single doses of trimethyltin X Cl (TMT) by the ip route. The effects of TMT administration were determined on lethality (3-6 mg/kg), spontaneous motor activity (SMA), and the physical appearance of the mice (0.3-3 mg/kg). The effects of TMT on lethality were strain dependent in that a single dose of 3 mg/kg, ip, produced approximately 35% lethality in the C57BL/6N strain during the first 72 hr following administration. Less than 15% lethality was observed at this dose in the BALB/c strain. In both strains, 3.5 mg/kg, ip, produced more than 70% lethality during the first 144 hr after administration. Higher doses produced proportionally greater lethality. The SMA of both strains was not affected significantly at doses below 1 mg/kg, ip. At 1 mg/kg a small decrease in activity was observed during the first 24 hr. At 3 mg/kg, SMA was initially decreased in both strains. However, the decrease was of smaller magnitude in the C57BL strain and was followed by a large increase in SMA which did not return to control levels for approximately 1 week. An increase in SMA was observed in the BALB/c strain on the fifth day following TMT but returned to control values by Day 6. At 3 mg/kg, ip, the C57BL mice were observed to have severe whole body tremors and were hypersensitive to external stimuli. The whole body tremor was not as marked in the BALB/c strain. Neuropathological studies on the treated mice indicated that the behavioral studies paralleled the pathology produced by TMT. These data confirm the initial observation of greater sensitivity of the mouse to toxic effects of TMT compared to the rat.     

Behavioral effects of trimethyltin in two strains of mice. II. Multiple fixed ratio, fixed interval

Adult male C57BL/6N and BALB/c mice were trained to respond under a multiple fixed-ratio 30, fixed-interval 600-sec schedule of milk presentation. After performance had stabilized, each mouse received a single dose of trimethyltin X Cl (TMT) by ip administration. Three hours after administration, behavioral testing was started and continued at 24-hr intervals thereafter. The behavior of mice receiving 0.3 mg/kg was unaffected at 3, 27, and 51 hr after administration. Likewise, no effects were seen at 3 and 27 hr after 1 mg/kg. However, at 51 hr after administration there was a significant change in the temporal patterning of fixed-interval responding in the C57BL/6N strain, as shown by a decrease in the fixed-interval quarter-life. There was no significant change in the BALB/c strain at this dose. At 3 mg/kg the responding of both strains was greatly affected, and responding remained disrupted for at least 6 to 7 weeks. Neuropathological examination of the brains of both strains of mice showed no significant lesions (light microscopy) at 0.3 and 1 mg/kg. At 3 mg/kg, severe neuronal necrosis was observed in the fascia dentata region in both strains. Thus, in mice receiving TMT, the behavioral deficits were closely paralleled by the presence or absence of significant neuropathology.     

In vitro benzo[a]pyrene metabolism from lindane-treated rat liver: effect of oral and acute administration, and comparison with phenobarbital and methylcholanthrene pretreatment.

Lindane (γ-hexachlorocyclohexane) was given orally to Sprague-Dawley male rats in their basal diet, at 24, 120, and 240 ppm, for 4 weeks. Acute intoxication was obtained with ip injection, at 20 mg/kg/day for 3 consecutive days, and the inductive effect was compared to phenobarbital (ip, 80 mg/kg/day for 3 days) and methylcholanthrene (ip, 20 mg/kg/day for 3 days) pretreatment. The spectrofluorometric measurement of arylhydrocarbon hydroxylase (AHH) activity did not show any induction after lindane and phenobarbital pretreatment, while the quantitative determination of benzo[a]pyrene (BP) metabolites by thin-layer chromatography indicated an increase of 3-hydroxy-BP (3-OH-BP). Differences in the BP/protein ratio could explain this discrepancy. Consequently, lindane appeared as a weaker inducer of AHH activity than phenobarbital. The in vitro binding of [¹⁴C]BP metabolites to DNA was largely enhanced by the oral administration of lindane from as low as 24 ppm in the diet and seemed dose related until 120 ppm, reaching two or three times the control value. The acute intoxication by lindane resulted in an increase in BP metabolite binding to DNA similar to that obtained with 120 ppm in the diet. From the chromatographic pattern of BP metabolites, it is concluded that lindane induced a large formation of 4,5-BP-dihydrodiol, which was related to a two-fold increase in epoxide-hydratase activity. From these results, lindane could be considered as a phenobarbital-like inducer.     

Studies on selenium-related compounds VI. Biosynthesis of dimethyl selenide in rat liver after oral administration of sodium selenate

Experiments were made to obtain data on the biological action of selenium in order to establish a standard for water quality for public water supply. Biosynthesis of dimethyl selenide in rat liver after oral administration of Na₂SeO₄ was investigated and the volatile selenium formed was identified. The study showed that dimethyl selenide, as a respiratory metabolite, was probably formed in the rat liver. Differences were noted as to dimethyl selenide formation from sodium selenite and sodium selenate in vitro. The test of single oral administration of sodium selenate indicated that dimethyl selenide formation increased progressively up to about 6 mg/kg and then reached a plateau at this dose. The increased accumulation of selenium in the liver after continuous oral administration was found to stimulate the methylation of selenium to dimethyl selenide. When sodium selenate was orally administered to rats, (CH₃)₂Se was found by TLC, GLC, and GC-mass spectrometry.     

The effect of age on salicylate-induced nephrotoxicity in male rats

The administration of 500 mg/kg sodium [14C]salicylate to 3- and 12-month-old male rats produced proximal tubular necrosis in the older animals but only mild nonspecific cellular changes in the younger group. The onset of renal damage was similar for both 3- and 12-month-old rats but recovery time was prolonged in the older rats. Covalent binding of salicylate equivalents was present in renal cortices from all rats and was largely confined to the mitochondrial fraction; however, older rats displayed five times more binding to this organelle than younger rats. Also the mitochondrial pathway for salicylurate synthesis was significantly inhibited in the older animals. These results demonstrate the existence of an age-dependent susceptibility to salicylate nephrotoxicity and suggest that mitochondrial injury may play an important role in the development of salicylate-induced proximal tubular necrosis.