Genotoxicity of N-methyl-N-nitrosourea in the presence of amphiphilic membrane-active compounds

With V79 Chinese hamster lung cells the dose-response relationship of sister chromatid exchange (SCE) induction by NMU shows a sigmoidal-shaped curve. In vitro, the time schedule of the short-lived (<60 min) SCE inducer (NMU) and the sister chromatid marker 5-bromodeoxyuridine (BrdU) addition to the cells has a significant effect on the SCE response suggesting a direct chemical and/or indirect biochemical interaction between both compounds. Certain amphiphilic molecules of a definite length, consisting of a hydrophobic chain and a polar end, have been shown to alter functions of various membrane integrated proteins by modifying membrane lipid structures. In vitro, three chemically distinct membrane-active compounds inhibit induction of SCEs following N-methyl-N-nitrosourea (NMU) treatment to different extents in a concentration-dependent manner. These findings with Chinese hamster lung cells confirm the results of in vivo experiments with bone marrow cells from Chinese hamsters. The anti-SCEs inducer effects are interpreted in terms of a decrease of nuclear bioavailability of NMU. Experimental results are not in favor of a competitive inhibition of SCE induction as a consequence of eventual alkylations of the amphiphilic molecules at their polar end. It is hypothesized that cell membrane structure modifications enhance the probability of chemical reactions of the strongly reactive SN₁ alkylating compound NMU with nucleophilic membrane sites. Membrane-active compounds with anti-SCE inducer capacities should be useful probes for the study of possible relationships between SCE induction and the carcinogenic as well as antitumor efficiency of NMU.     

Effect of reduced glutathione (GSH) on pharmacokinetics and distribution of rifamycin SV in rats

Changes of the pharmacokinetic parameters of rifamycin SV in the blood and changes of the antibiotic concentration in rat organs after its coadministration with glutathione in reduced form (GSH). in oxidized form (GSSG) and with the individual amino acids entering the tripedtide were studied. The use of reduced glutathione together with rifamycin was found to accelerate the distribution phase in the kinetics of the antibiotic in the blood by acceleration of transport of the drug from central compartment (blood) to tissue compartment (tissues), this being reflected by increased K₁₂ and K₁₂/K₂₁ values. Studies of the distribution of rifamycin in individual rat organs have shown GSH to significantly elevate the concentration of the antibiotic in the lungs with concomitant reduction of its level in the liver. The blocking of free sulfhydryl groups of glutathione by its use in oxidized form completely abolished this effect. The administration of rifamycin together with l-cysteine resulted in increased concentration of the antibiotic in both lungs and liver. The two other amino acids entering GSH —glycine and l-glutamic acid — did not cause any changes of rifamycin concentration in the organs. From these results it may be concluded that the mechanism of the observed effect of glutathione on rifamycin concentration differs in the lungs and liver though both effects are mediated by the free sulfhydryl groups of glutathione.     

On the metabolism of allopurinol. Formation of allopurinol-1-riboside in purine nucleoside phosphorylase deficiency

Allopurinol-1-riboside, a major metabolite of allopurinol, is commonly thought to be directly synthesized by purine nucleoside phosphorylase (PNP) in vivo. As this enzyme is otherwise believed to function in vivo primarily in the direction of nucleoside breakdown, we have determined by high performance liquid chromatography and a conventional chromatographic method the urinary metabolites of allopurinol in a child deficient of PNP. In this patient approximately 40% of urinary allopurinol metabolites consisted of allopurinol-1-riboside, thus proving the possibility of indirect formation of allopurinol-1-riboside via allopurinol-1-ribotide in vivo, catalysed by hypoxanthine guanine phosphoribosyltransferase (HGPRT) and a phosphatase.     

Alteration of hepatic microsomal enzymes by griseofulvin treatment of mice.

Feeding mice a diet containing 2.5% griseofulvin (GF) for 12 days caused an increase in liver weight to 9 per cent of the body weight with a proportional decrease in microsomal protein/g of liver wet weight. With respect to microsomal protein, the cytochrome P-450 content was 50 per cent and cytochrome b₅ was 200 per cent of that in control mouse liver microsomes. The amount of increase in cytochrome b₅ was approximately the same as the amount of decrease in cytochrome P-450. and there was no change in the total microsomal heme content. The microsomal content of NADH-cytochrome b₅ reductase, as measured by ferricyanide reduction, was unchanged, but in agreement with the elevated cytochrome b₅ content, NADH-cytochrome c reductase activity was doubled. While the cytochrome P-450 level was low in microsomes after GF feeding, the NADPH-cytochrome c reductase was significantly elevated. Since this enzyme is generally considered to be rate limiting for many mixed function oxidase reactions, its increase may explain the normal to slightly elevated rates of metabolism in vitro of several type I and type II substrates. Although cytochrome b₅, has been suggested as being rate limiting for input of a second electron to cytochrome P-450 linked mixed function oxidations, elevation of cytochrome b₅ in the microsomes did not change the extent of NADH-synergism of NADPH-supported aminopyrine demethylation. NADH-supported Δ,10-fatty acid desaturase activity, which requires cytochrome b₅, was elevated several-fold after GF feeding. In contrast, NADPH-supported lipid peroxidation showed a slower onset after GF treatment; the NADH-supported reaction, however, was slightly elevated.     

Effect of hexachlorobenzene on the activities of hepatic alcohol metabolizing enzymes

To study the effect of experimental hepatic porphyria on the activities of hepatic alcohol metabolizing enzymes, female rats received a chow diet containing 0.05% hexachlorobenzene (HCB). After long-term HCB treatment for 60 days hepatic porphyria developed as evidenced by increased hepatic delta-aminolevulinic acid synthase activity and enhanced urinary excretion of delta-aminolevulinic acid, porphobilinogen and total porphyrins. Concomitantly, the activities of the hepatic microsomal ethanol oxidizing system (MEOS) were strikingly augmented by 213% (P less than 0.05) and 177% (P less than 0.01) when expressed per g of liver wet weight or per 100 g of body weight, respectively, whereas hepatic alcohol dehydrogenase activities remained virtually unchanged. Moreover, hepatic catalase showed only a trend for a slightly lower enzymic activity under these experimental conditions. The present data therefore show that experimental hepatic porphyria is associated with alterations of hepatic MEOS activities, which in turn may be a factor for the manifestation of human hepatic porphyrias in the course of alcohol consumption.     

Influence of muzolimine on arterial wall elastin

Muzolimine, 3-amino-1-(3,4-dichloro-alpha-methylbenzyl)-2- pyrazolin -5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60-300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolimine for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastin under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.     

Exhalation of acetone by rats on exposure to trans-1,2-dichloroethylene and related compounds

If rats are exposed in a closed system to trans-1,2-dichloroethylene (900 ppm initial concentration), acetone is exhaled at a rate of 1.95 ± 0.79 (SD)$\text{μmol}\text{h}$ per kg body weight. Acetone exhalation may reflect metabolic changes induced by the xenobiotic, and can also be elicited by exposure to cis-1,2-dichloroethylene, perchloroethylene and vinylidene fluoride.     

Stimulatory and inhibitory effects of manganous and ferrous ions on epinephrine oxidation.

Low concentrations of manganous ions stimulate the oxidation of epinephrine to adrenochrome by rat liver microsomes, or by isolated NADPH-cytochrome c reductase, 4 fold. In contrast, much higher manganous ion concentrations are required for the chemical oxidation of epinephrine. Whereas the microsomal manganese stimulated epinephrine oxidation is inhibited by Superoxide dismutase, as is the nonstimulated enzymatic oxidation, Superoxide dismutase has no inhibitory effect on the oxidation of epinephrine in the absence of enzymes.Ferrous ions inhibit the manganous ion stimulated chemical oxidation of epinephrine more effectively than the similarly stimulated enzymatic oxidation of epinephrine. Based upon these observations different mechanisms for chemical and enzymatic manganous ion-induced epinephrine oxidation are suggested. Furthermore, possible implications of the metal ion-stimulated or inhibited oxidation of catechols in vivo are discussed.     

Inhibition of CBrCl3-induced lipid peroxidation in rats in vivo by (+)-cyanidanol-3

(+)-Cyanidanol-3 given to rats in vivo decreased the ethane production induced by treatment with 100 mg CBrCl₃/kg. The extent of this inhibitory effect was dependent on the dose of (+)-cyanidanol-3. It is concluded that (+)-cyanidanol-3 prevents the lipid peroxidation induced in vivo by CBrCl₃.     

The influence of isotonic saline administration on the urinary excretion of kallikrein in rats

Acute saline loading is known to increase kallikrein excretion. To clarify whether this is a specific stimulatory effect or rather a non-specific wash-out, pentobarbital anesthetized rats were loaded thrice (5 min infusions) at 40 min intervals with a volume of 150 mM NaCl equal to 5% of their body wt. The effect of such a load on the central venous pressure was studied in a separate group of rats. Control animals did not receive the infusions. Kallikrein excretion (amidolytic assay) increased with the first, but decreased with the subsequent saline administrations. The rise observed after the first load lost significance when kallikrein excretion was related to that of creatinine. The reduction observed after the second and third infusions remained significant even when expressed per mg of creatinine. Thus, saline load induced kallikrein "stimulation" is due to a non-specific wash-out. Similar transient enhancements of central venous pressure were observed after each of the three loads. This, together with the unchanged creatinine excretion (except for the rise seen after the first load) indicate that the lack of kallikrein stimulation after the second and third loads was not due to the appearance of heart failure. Saline loaded rats had a renal kallikrein activity at the end of the experiment which did not differ from that of controls. Plasma aldosterone concentration was reduced in saline infused rats, and it correlated with the kallikrein excretion when both, NaCl loaded and control rats, were taken into account.     

Activation of the O-glucuronide of the carcinogen N-hydroxy-N-2-fluorenylacetamide by enzymatic deacetylation in vitro: formation of fluorenylamine-tRNA adducts.

The O-glucuronide of N-hydroxy-N-2-fluorenylacetamide (N-GIO-FAA) was deacetylated by guinea pig liver. tRNA reacted with the product of this deacetylation, the O-glucuronide of N-2-fluorenylhydroxylamine (N-GIO-FA), to give fluorenylamine-substituted nucleic acid adducts. The quantity of adduct formation was used to determine deacetylase activity.Of the various guinea pig tissues assayed, only the liver contained enzyme activity, and this activity was confined to the microsomal fraction of the cell. Guinea pig liver microsomes were about four times as active as rabbit liver microsomes and about fourteen times as active as rat liver microsomes in promoting fluorenylamine-tRNA adduct formation. Adduct formation induced by guinea pig microsomes was about seven times greater at pH 8.5 than at pH 7.0.The aglycone of the O-glucuronide, N-hydroxy-N-2-fluorenylacetamide (N-hydroxy-FAA) also yielded flourenylamine-substituted nucleic acid adducts following deacetylation at pH 8.5 by guinea pig liver microsomes in the presence of tRNA. In contrast to resuls obtained with N-GIO-FAA, adduct formation with N-hydroxy-FAA was not as efficient, and it was independent of pH over the range 7.0–8.5. Rabbit and rat liver microsomes were more active in promoting adduct formation of tRNA with N-hydroxy-FAA than with N-GIO-FAA.The differential inhibition of the microsome-induced formation of adducts of N-GIO-FAA and N-hydroxy-FAA with tRNA affirms that the first step in the binding mechanism of N-GIO-FAA with tRNA is enzymatic deacetylation and not hydrolysis to the aglycone N-hydroxy-FAA.     

Inductive effect on hepatic enzymes and acute toxicity of individual polychlorinated dibenzofuran congeners in rats

Toxicological assessment of 13 individual polychlorinated dibenzofurans (PCDFs) with varying chlorine substitutions was made in young male Wistar rats. All the 9 congeners having at least three chlorine atoms in the ring positions at 2, 3, 7 and 8, such as 1,2,7,8-, 2,3,6,7-, and 2,3,7,8-tetrachlorodibenzofurans (TCDFs), 1,2,3,7,8-, 1,2,6,7,8-, 2,3,4,7,8-pentachlorodibenzofurans (PenCDFs), 1,2,3,4,6,7- and 1,2,3,4,7,8-hexachlorodibenzofurans (HCDFs), exhibited a typical 3-methylcholanthrene (MC)-type induction, i.e., a marked increase in activity of aryl hydrocarbon hydroxylase (AHH) and DT-diaphorase in the liver. The most potent congeners were 2,3,7,8-TCDF and 2,3,4,7,8-PenCDF, both of which significantly induced the AHH and DT-diaphorase even at a single dose of 1 μg/kg. On the contrary, the congeners having two or less chlorine atoms in the lateral ring positions, such as 2,8-dichlorodibenzofuran, 1,3,6,7- and 1,3,6,8-TCDFs, and 1,2,4,6,8-PenCDF, did not show any inductive effect on these hepatic enzymes at the single dose of 5 or 10 mg/kg. All the MC-type PCDFs except for 1,2,7,8-TCDF caused a marked atrophy of the thymus and a hypertrophy of the liver in rats, while no toxic signs were observed in animals treated with the congeners lacking the MC-type inducing ability. The ranking of toxic potencies of the MC-type PCDFs coincided well with their inducing abilities. The hepatic disposition of the congeners varied markedly. For example, 2,3,7,8-TCDF and 2,3,4,7,8-PenCDF showed an equipotent toxicity, however, the hepatic concentration of PenCDF was about 20-fold higher than that of TCDF. These results clearly indicate that the acute toxicity of PCDF congeners is well correlated with their MC-type inducibility, but not with their hepatic distribution.     

Influence of highly unsaturated phosphatidylcholine on the effects of ouabain and some cardioactive drugs on cardiac contractile force and Na+, K+-ATPase activity.

Isolated left guinea pig auricles and cardiac Na,K-ATPase from calf heart were incubated with highly unsaturated phosphatidylcholine (PC) for 2 hr. Thereafter the actions of ouabain on Na, K-ATPase, and of ouabain, digoxin, digitoxin, isoproterenol, acetylcholine, pentobarbital and different extracellular Ca²⁺ concentrations on contractile force of the auricles were investigated. PC itself altered neither the ATPase activity nor the intracellular ionic homeostasis, nor contractile force of the auricles. Similarly, the staircase phenomenon, the contractile response to different extracellular Ca²⁺ concentrations, to pentobarbital and ouabain, and the extent of ouabain-induced inhibition of the ATPase were not influenced. In addition the toxicity of ouabain was not altered. However, the rate of development of the ouabain-induced ATPase inhibition, as well as of the positive inotropism and toxic effects of ouabain, digoxin and digitoxin was markedly reduced. The maximum inotropic effect of digoxin and digitoxin, and the toxicity of digitoxin were significantly enhanced. Remarkably, the binding of [³H]digitoxin was significantly diminished in PC-pre-incubated auricles, binding of [³H]ouabain, however, remained unchanged. The dose-response curves to isoproterenol and acetylcholine, the latter also in the presence of physostigmine, were markedly parallel-shifted to the right. The modifying effect of highly unsaturated PC on the action of the drugs studied is suggested to be due to an altered physico-chemical state (fluidity) of the outer surface of the cellular membrane.     

Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure

Timed-pregnant rabbits were administered a single, sc injection of cadmium chloride (0, 0.125, 0.25, or 0.50 mg cadmium/kg body wt) 48 hr prior to being killed at term. Although the doses chosen were below that shown to produce significant fetal lethality, the 0.50 mg/kg dose did produce a significant reduction in fetal body weight, liver weight, kidney weight, and crown-rump length while having no effect on placental weight. Fetal hepatic metallothionein levels were significantly increased at all three doses; however, a significant increase in metallothionein zinc content was seen only at the two higher doses. Metal distribution studies indicated that the increase in fetal metallothionein and the accompanying increase in cytosolic zinc coincided with an apparent redistribution of the metal from extrahepatic sites to the liver. The whole fetal content of zinc was not significantly altered by the maternal administration of cadmium.     

Circular dichroism studies on the interaction of four structurally related long-acting sulfonamides with human and bovine serum albumin

The interaction of four sulfonamides with bovine and human serum albumin (BSA and HSA) was investigated by means of circular dichroism and u.v. absorbance measurements. In the case of all sulfonamides, extrinsic Cotton effects could be found for the interaction with BSA and HSA. The anisotropy factors of the electronic transitions in the p-amino benzenesulfonic acid and in the heterocyclic moieties of the drugs do not differ, therefore it is concluded that both parts of the sulfonamide molecules, the p-amino benzenesulfonic acid part and the heterocyclic ring are bound to the albumin surface. The circular dichroism measurements especially reveal some differences of the interaction of the sulfonamides with both albumins.     

Effect of short-term ozone exposure on exogenous thyroxine levels in thyroidectomized and hypophysectomized rats

Short-term ozone exposure (1 ppm × 24 hr) of male rats results in a significant reduction of circulating thyroid hormones and thyroid stimulating hormone (TSH). The reduction of thyroid hormone levels after ozone exposure has been hypothesized as a possible adaptive mechanism to enhance survival of rats during ozone exposure. In this study, we investigated the effect of ozone on thyroid hormone (T₄) levels in thyroidectomized and hypophysectomized rats which received exogenous T₄ in the drinking water. Groups of normal, intact rats, thyroidectomized rats maintained on T₄ at doses ranging from 75 to 1000 μg/liter, and hypophysectomized rats maintained on 300 μg T₄/liter were exposed to ozone (1 ppm × 24 hr). Plasma T₄ concentrations were significantly reduced after ozone exposure, and the results indicated that the higher the circulating T₄ levels before exposure the more they were reduced after ozone exposure. This reduction in T₄ levels cannot be accounted for in these animals by reduced pituitary TSH levels or the effects of fasting, but is likely to be due to peripheral changes in plasma thyroid binding proteins initiated by ozone exposure.     

Turnover kinetics of dopamine and norepinephrine in the forebrain after kindling in rats.

Turnover rate constants for dopamine (DA) and norepinephrine (NE) were estimated for the forebrain of kindled rats from which overt motor seizures had been elicited by stimulation of basolateral amygdala. Kindled rats had significantly higher rate constants for DA turnover in the hemisphere ipsilateral to electrical stimulation. Turnover rate constant for DA in contralateral hemisphere tended to be greater in kindled rats and was not significantly different from that determined for ipsilateral hemisphere. NE turnover rate constants were not significantly different between hemispheres nor between groups of kindled and control rats.     

Enhancement of mutagenic activity in Salmonella by contraceptive steroids

Two oral contraceptive steroids, mestranol and norethynodrel, were evaluated for mutagenicity in the Salmonella histidine reversion assay. The pure forms of the hormones were not mutagenic when tested with either missense (TA1535, TA100) or frameshift (TA98, TA1538, TA1537) strains. In vitro activation of the hormones with liver homogenates from rats induced either with phenobarbital or Aroclor did not influence these results. However, mestranol was capable of enhancing the mutation yield obtained by an ineffective subthreshold dose of 2-acetylaminofluorene. Dimethyl sulfoxide extracts of two contraceptive pills, Ovulen-21 (containing mestranol) or Enovid-E (containing mestranol or norethynodrel), also were nonmutagenic. But again, both these extracts were capable of enhancing the mutation yield induced with an ineffective dosage of 2-acetylaminofluorene and N-nitrosopiperidine. These studies point to the possible promotional effect and subsequent potential hazard to the female consumers who use these hormones as a means of pregnancy control.     

Altered lipid metabolism in 2,5-hexanedione-induced testicular atrophy and peripheral neuropathy in the rat

2,5-Hexanedione (HD) induces testicular atrophy and peripheral neuropathy in rats. Since altered lipid metabolism is frequently associated with these disease states, lipid metabolism was investigated in vitro in testes and sciatic nerves of rats fed 1% HD in the drinking water for 6 weeks. Testes from HD-treated rats were 30–60% smaller and weighed threefold less than testes from pair-fed control (PFC) rats. Compared to testes from PFC rats, testes from HD rats exhibited increased incorporation of [¹⁴C]acetate into phospholipids (344%), triacylglycerols (281%), and cholesteryl esters + hydrocarbons (246%) but decreased incorporation into free fatty acids (25%) and sterols + diacylglycerols (65%). The increased incorporation of [¹⁴C]acetate into phospholipids induced by HD reflected an approximate 300% increase into phosphatidyl choline, lysophosphatidyl choline, phosphatidyl serine + phosphatidyl inositol, and phosphatidyl ethanolamine and a disproportionate 800% increase into sphingomyelin. HD rats exhibited clinical signs of peripheral neuropathy, including everted and flat foot placement and hindlimb weakness; similar changes were not observed in PFC rats. In sciatic nerves, the incorporation of [¹⁴C]leucine was decreased into sterols + diacylglycerols (47%), digitoninprecipitable sterols (45%), and cholesteryl esters + hydrocarbons (40%) in HD compared to PFC rats; incorporation of [¹⁴C]leucine into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. In contrast to the testis and nerve, lipid metabolism in the liver was similar in HD and PFC rats. The concentrations of 2,5-hexanedione and 2,5-dimethylfuran, respectively, were 0.6 ± 0.3 and 6.5 ± 0.9 μg/g wet weight in the testes and 3.1 ± 0.4 and 3.0 ± 0.4 μg/g wet weight in the livers of HD rats. The data indicate that altered metabolism is associated with HD-induced testicular atrophy and distal axonopathy.