Toxicological studies on 5-methoxymethyl-2′-deoxyuridine,a new antiviral agent

Toxic effects of 5-methoxymethyl-2′-deoxyuridine (MMUdR) were studied in White Swiss mice. Pathological, hematological, and clinical chemistry parameters were examined. No morbidity or mortality was observed after administration of MMUdR in a single dosage of 4000 mg/kg and in repeated dosages of 1000 mg/kg daily for 15 days. No gross or histopathological lesions related to MMUdR treatment were observed in the tissues examined from mice of the acute toxicity studies. Histopathological lesions were found in liver and intestine of mice treated with 1000 mg/kg/day for 15 days. These lesions consisted of cytoplasmic vacuolation of hepatocytes and atrophy of intestinal villi. The latter was associated with dilation of the intestine. Control livers contained significantly more mitotic figures than did those of treated animals. The mean corpuscular volume (MCV) of treated mice (4000 mg/kg) was significantly lower than that of controls. After repeated low doses, treated animals exhibited a hemoconcentration effect as evidenced by a lower packed cell volume and MCV and a higher mean corpuscular hemoglobin concentration and total solids in serum. Microscopic examination of marrow smears from control and treated animals from both acute and subacute toxicological studies demonstrated normal hematopoiesis. Differences in serum alkaline phosphatase and serum glutamic oxaloacetic transaminase activities between treated and control mice were observed in the subacute toxicity study. No toxic effect upon fetuses and no teratogenic effects were observed in 152 offspring from mice receiving 500 mg/kg/day of MMUdR for 19 or 21 days.     

Altered ontogeny of glutathione S-transferases by 2,4,5-2',4',5'-hexachlorobiphenyl.

Date-bred rats were treated orally from the 8th through the 18th day of gestation with pure congeners of polychlorinated biphenyls (PCBs). At designated stages of postnatal development offspring of treated dams were sacrificed and glutathione S-transferase (G S-t) activity levels were determined using the substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). Male and female rats exposed perinatally to 2,4,5-2′,4′,5′-hexachlorobiphenyl (6-CB) had higher levels of G S-t than the controls at all ages examined (Days 6, 21, 55 postpartum). Treatment of adult male rats with increasing doses of 6-CB revealed that glutathione conjugation toward CDNB could be more easily enhanced than toward DCNB. Pure 6-CB added to control cytosol did not change G S-t activities. Mixtures of cytosol from 21-day-old control and 6-CB animals had activities similar to the calculated mean average of the two.     

Mechanism and kinetics of the inhibition of dopamine-beta-hydroxylase by 2-mercaptoethylguanidine

The inhibition of dopamine-β-hydroxylase (DBH) by 2-mercaptoethylguanidine (MEG) was studied using a partially purified DBH preparation. The MEG-induced inhibition of DBH decreased progressively with increasing Cu²⁺ concentration; equal concentrations of MEG and Cu²⁺ were without effect, supporting the hypothesis that the mechanism of inhibition is through binding of enzymic Cu. It was shown kinetically that MEG inhibits purified DBH by a two-inhibitor molecule interaction. Since MEG inhibits DBH by binding enzymic Cu, two Cu ions must be available at the active site for binding with the two MEG molecules. Several congeners of MEG and other compounds were used to study the structure-activity relationship (SAR) of the MEG inhibition of DBH. The SAR correlated with a hypothetical model of the enzyme active site based on the information obtained from the kinetic studies of the MEG inhibition of DBH. The structure-activity relationship suggested that the intramolecular distance between the anionic site and the site of β-hydroxylation of the substrate, dopamine (DA), was coincident with the intramolecular distance between the positive charge of the inhibitor and the site of binding of the Cu of DBH.     

Effects of 2-mercaptoethylguanidine and other compounds on norepinephrine synthesis by adrenal medullary granules

The effects of 2-mercaptoethylguanidine and other compounds on dopamine uptake and norepinephrine synthesis by adrenal medullary granules were studied. The effects of these materials on crude dopamine-β-hydroxylase were also tested. A microanalytical technique was developed which allows the use of the natural precursor, dopamine, in studies of uptake and synthesis by adrenal medullary granules. The conversion of dopamine to norepinephrine by dopamine-β-hydroxylase was also studied with this technique. Catecholamines were assayed as their dansyl (5-dimethylaminonaphthalene-1-sulfonyl) derivatives, thereby increasing greatly the resolution and sensitivity of detection. The effects of 2-mercaptoethylguanidine and reserpine on dopamine uptake and norepinephrine synthesis by medullary granules were compared. Reserpine inhibited norepinephrine synthesis indirectly through inhibition of dopamine uptake. 2-Mercaptoethylguanidine, on the other hand, depressed norepinephrine synthesis in intact granules by a direct inhibitory effect on dopamine-β-hydroxylase rather than on the uptake mechanism. 2-Mercaptoethylguanidine increased the rate of norepinephrine synthesis by crude dopamine-β-hydroxylase in the presence of added Cu²⁺ and inhibited norepinephrine synthesis in the absence of added Cu²⁺. The nature of the latter effect was resolved by demonstrating the formation of a Cu-2-mercaptoethyl-guanidine complex in pure solutions. Since dopamine-β-hydroxylase is a Cu-containing enzyme, the mechanism of the 2-mercaptoethylguanidine inhibition of dopamine-β-hydroxylase appears to be through the binding of enzymic Cu.     

Interactions of S-(2-haloethyl)-mercapturic acid analogs with plasmid DNA

A series of related S-(2-haloethyl)-L-cysteine analogs were synthesized and their interaction with DNA was studied with plasmid pBR322. Both S-(2-chloroethyl)-L-cysteine (CEC) and S-(2-bromoethyl)-L-cysteine (BrEC) rapidly induced relaxation of the supercoiled plasmid as determined by agarose gel electrophoresis and electron microscopy, whereas S-(2-fluoroethyl)-L-cysteine did not interact with DNA. The relaxation was most probably due to strand scission at alkylated labile sites in the DNA. When 35S-labeled CEC or BrEC was used as the substrate, covalent binding of 35S to DNA was obtained; CEF displayed a somewhat higher binding than BrEC. No binding of 35S was obtained with (2-hydroxyethyl)-L-[35S]cysteine, [35S]cysteine, or [35S]cystine, substrates which did not induce relaxation of the DNA. Esterification of the carboxyl group resulted in a somewhat lower rate of DNA strand scission, whereas N-acetylation prevented the cysteine analogs from inducing DNA strand breaks. S-(2-Chloroethyl)-glutathione (GSH) did not interact with DNA as determined by lack of effect on the superhelicity of DNA, a finding which is in agreement with the hypothesis that the primary amine groups of CEC or BrEC may participate in the formation of reactive intermediates which can interact with DNA. S-(2-Hydroxyethyl)-GSH and S-(2-hydroxyethyl)-L-cysteine were unable to induce DNA strand breaks. Neutral denaturation of supercoiled pBR322 treated with the analogs revealed that compounds which were able to induce DNA strand breaks also interfered with denaturation of double-stranded circular DNA. No such interference was observed when double-stranded linear DNA (obtained by BamH1 restriction digestion) was treated with the analogs prior to denaturation. These data indicate that a marked difference exists between S-(2-chloroethyl)-L-cysteine and S-(2-chloroethyl)-glutathione in their reaction with supercoiled plasmid DNA. Either a major difference exists in the reactivity of the corresponding episulfonium ions of these conjugates or a separate mechanism of alkylation based on a free alpha-amino of the cysteine conjugate is participating in DNA strand breakage and possible crosslinking. In vivo toxic effects of these S-(2-chloroethyl) conjugates are predicted to be distinctly different.     

Acute toxicity of conventional versus shale-derived JP5 jet fuel: light microscopic,hematologic, and serum chemistry studies

Rats were gavaged with 60, 48, 38, 30, or 24 ml/kg body wt of either petroleum-derived JP5 jet fuel or one of three samples of shale-derived JP5 jet fuel. The surviving rats were killed at 14 days after dosing. In another study, rats were gavaged with one of three shale-derived fuels at the rate of 24 ml/kg body wt and killed at 1, 2, or 3 days postdosing. There was a significant difference in lethality of the three shalederived fuels, even though all originated from the same shale deposit. The difference in toxicity was attributed to variations in the chemical composition of the crude oil in refining processes. Elevated packed cell volume and red cell count on Days 1 and 2 indicated severe dehydration. White cell counts were markedly reduced on Days 1 and 2. All hematologic parameters were similar to control values by 3 days postdosing. Pathologic findings in rats that died within 24 hr of dosing indicated cardiovascular collapse. Rats that died later exhibited hepatic periportal fatty change and renal hyaline droplet formation and fatty change. Rats sacrificed at daily intervals following administration of 24 ml/kg JP5 had moderate renal and hepatic functional alterations, as indicated by serum chemistry determinations. Renal hyaline droplet formation and fatty change was microscopically evident on all 3 days, but hepatic fatty change was not present until Day 2.     

The transfer of 2,4,5,2',4',5'-hexachlorobiphenyl to fetuses and nursing offspring. I. Disposition in pregnant and lactating mice and accumulation in young

The disposition of 2,4,5,2′,4′,5′-hexachlorobi[¹⁴C]phenyl (6-CB) in pregnant and lactating mice and its transfer to fetuses and nursing offspring were examined by administration of 6-CB to virgin female mice 2 weeks prior to mating. No differences were observed in tissue concentrations of [¹⁴C]6-CB in pregnant animals when compared to virgin controls. However, on the day of birth, liver, adipose tissue, and kidney 6-CB contents in pregnant mice were higher than those in virgins sacrificed concurrently. Transplacental passage of 6-CB was minimal. Lactating mothers eliminated essentially their entire body burden of the PCB by 20 days postpartum and 6-CB was, in turn, accumulated by nursing offspring. No measurable elimination of 6-CB was observed from virgin controls during this time. Thus, although little passage of 6-CB occurred across the placenta once it had been sequestered into storage depots, it was readily transferred to suckling offspring through the lactating mammary gland.     

The mutagenicity of some of the proposed metabolites of direct black 38 and pigment yellow 12 IN THE Salmonella typhimurium assay system

The Ames Salmonella/mammalian microsome assay system was used to evaluate the mutagenic potential of some of the proposed metabolites of Direct Black 38 and Pigment Yellow 12. Direct Black 38, benzidine, 4-aminobiphenyl, monoacetylbenzidine, and diacetylbenzidine were mutagenic for at least one of the tester strains after metabolic activation with mouse liver S-9 fractions. The proposed metabolites of Pigment Yellow 12, namely dichlorobenzidine, monoacetyldichlorobenzidine and diacetyldichlorobenzidine were strongly mutagenic for Salmonella typhimurium TA 98.The major metabolite in the urine from hamsters given a single dose of Direct Black 38 (100 mg/kg) was found to be monoacetylbenzidine. Monoacetylbenzidine and the urine from the animals treated with Direct Black 38 were active in the Ames test only after S-9 fraction was added to the test mixture.     

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.     

Dose-response relationships in 1,3-butanediol-induced potentiation of carbon tetrachloride toxicity

Pretreatment of rats with 1,3-butanediol (BD) (1.0, 5.0, or 10.0% in drinking water) for 7 days enhanced the hepatotoxic, but not the nephrotoxic, effect of a single dose of CCl₄ (0.1 ml/kg, ip) in a dose-related manner. Biochemical tests and light microscopy were employed to assess toxicity. The smallest dosage of BD needed to potentiate CCl₄ hepatotoxicity was estimated to be between 0.1 and 1.0 g/kg/day. The potentiated response appeared to be related to the severity of the metabolic ketosis produced by BD. Pretreatment with BD (10.0%) resulted in increased hepatic microsomal activity and cytochrome P-450 content; the in vitro binding of ¹⁴CCl₄-derived radioactivity to microsomal protein was increased both aerobically and anaerobically, but was greater under aerobic conditions. BD appeared to potentiate CCl₄ liver injury, at least in part, by producing an increase and/or alteration in mixed function activation of CCl₄ to toxic metabolites. Pretreatment with BD (1.0, 10.0%) also resulted in a reduction of hepatic glutathione content. Thus, BD potentiation may arise through several, interrelated pathways.     

Comparison of the enzymatic and colchicine binding properties of subcellular fractions of rat brain

Capacity for colchicine binding and activities of lactate dehydrogenase, monoamine oxidase and cholinesterase were determined in subfractions of adult rat brain. Colchicine binding activity was mainly found in cytoplasmic fractions. The fractions containing membranes bound little colchicine. Activity and specific activity of colchicine binding were roughly comparable with the corresponding values of lactate dehydrogenase. Colchicine binding in particulate fractions therefore is probably due to contamination with cytoplasmic protein. Polyacrylamide gel electrophoresis revealed some similarity between the protein patterns of the subsynaptosomal particulate fractions and showed that colchicine binding protein is not present in synaptic vesicles.     

Epoxide hydrase in human liver biopsy specimens: assay and properties

A sensitive assay for the determination of epoxide hydrase activity in needle biopsy specimens of human liver has been developed with [³H]styrene oxide as substrate and was used for the study of some properties of human epoxide hydrase. Levels of epoxide hydrase in liver of man are 4.71 ± 0.41 nmoles styrene glycol/mg protein per min, comparable to guinea-pig (5·00 ± 0·38) rather than to Rhesus monkey (13·16 ± 0·88). Human hepatic epoxide hydrase which was found exclusively in the microsomal fraction, was solubilized with Cutscum, a neutral detergent, and purified. The enzyme was remarkably stable as long as it was particle-bound. Neither dialysable cofactors nor endogenous activators or inhibitors appeared to exist in the homogenate. The optimum pH for the purified enzyme was 9. The apparent K_m was 0.38 mM and the apparent V_max was 62.1 nmoles product/mg protein per min with respect to styrene oxide as the substrate. The product, styrene glycol, had no inhibitory effects. High (5–17 mM) concentrations of substrate, styrene oxide, markedly inhibited epoxide hydrase activity at low (0·2 mg/ml) but not at high (2 mg/ml) concentrations of protein, indicating that this inhibition may be due to the alkylating properties of the subsirate, styrene oxide. Sulfhydryl reagents slightly but significantly inhibited the enzyme suggesting that no sulfhydryl group is essentially involved in the catalytic mechanism at the active site, but that sulfhydryl group(s) may be of importance for holding the enzyme molecule in the optimal conformation for maximal activity. Chelating agents, a carbonyl reagent and β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) had no effect.     

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.     

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.     

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.     

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.     

Subchronic oral toxicology of 4-fluoro-3-nitroaniline in the rat

4-Fluoro-3-nitroaniline (4-F-3-NA), an intermediate used in the production of commercial hair dyes, was administered daily for 90 days via po intubation to Charles River CD rats at doses of 40, 120, or 360 mg/kg to determine its subchronic toxicology. All animals receiving 360 mg/kg died prior to termination of the study. The principal effect to rats of 4-F-3-NA exposure was alteration of the hematopoietic system characteristic of hemolytic anemia. Hematocrit, hemoglobin concentration, and erythrocyte count were decreased and reticulocytes increased in a dose-related pattern in both male and female animals. Histopathological change consistent with hemolytic anemia was observed in the bone marrow (erythroid hyperplasia), kidneys (deposition of biliverdin and pigment-laden macrophages in the tubules), liver (pigmented Kupffer cells), and spleen (increased hematopoiesis) in males and females. Heinz bodies and an increase in methemoglobinemia were not observed in this study. In the testes, aspermia and testicular degeneration were seen in isolated tubules, but these changes were significant only at the high dose. Additional pathologic changes noted at the high dose included colloid depletion and follicular cell hypertrophy of the thyroid gland, hypertrophy of the zone fasciculata in the adrenal glands, and lymphoid cell depletion in mesenteric lymph nodes. The calcification of the myocardium and coronary arteries was considered metastatic in nature, secondary to kidney damage. Serum ALT and BUN were elevated in males and AP was elevated in females from the high dose group. Glycosuria, bilirubinuria, and urobilinogenuria were also increased in both sexes at this dose.     

Disposition of radiolabeled benzoquinolizinyl propionanilide hydrochlloride in rats and dogs and identification of several of its metabolites

The biological disposition of ¹⁴C-labeled N-(1,3,6,7-hexahydro-11bH-benzo(a)quinolizin-2-yl) propionalide hydrochloride (1), an experimental hypertensive agent was studied in the rat and dog. Both species received a single po dose of [¹⁴C]I, 31.6 mg/kg to rats and 6 mg/kg to dogs. The compound was well absorbed by both species. The maximum plasma level of undifferentiated ¹⁴C in the dog was nearly equal to that in rat plasma, despite the fivefold dose difference. Plasma ¹⁴C levels in the dog were more persistent: biological half-fife estimates (α phase) were 5.9 and 1 hr for the dog and rat, respectively. In the rat the highest tissue ¹⁴C concentrations were found in liver and the lowest were found in brain. In both rats and dogs, more than 80% of the administered radioactivity was recovered from feces and urine within 96 hr, mostly within the first 24 hr. The 96-hr urinary: fecal excretion ratios of ¹⁴C were 35:65 in both species. Metabolic end products of [¹⁴C/³H]I (50 mg/kg, rat and 30 mg/kg, dog) were the same in both species. It was noted that quantitatively the rat excreted two to three times as much glucuronic acid-conjugated material as the dog.