Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.     

Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretion

Estradiol secreted by the maturing follicle is the primary trigger for the surge of gonadotropins leading to ovulation. Progesterone has stimulatory or inhibitory actions on this estrogen-induced gonadotropin surge depending upon the time and dose of administration. The administration of progesterone to immature ovariectomized rats primed with a low dose of estradiol induced a well-defined LH surge and prolonged FSH release, a pattern similar to the proestrus surge of gonadotropins. A physiological role of progesterone is indicated in the normal ovulatory process because a single injection of the progesterone antagonist RU 486 on the day of proestrus in the adult cycling rat and on the day of the gonadotropin surge in the pregnant mare's serum gonadotropin stimulated immature rat resulted in an attenuated gonadotropin surge and reduced the number of ova per ovulating rat. Progesterone administration brought about a rapid LHRH release and an decrease in nuclear accumulation of estrogen receptors in the anterior pituitary but not the hypothalamus. The progesterone effect was demonstrated in vitro in the uterus and anterior pituitary and appears to be confined to occupied estradiol nuclear receptors. In in vivo experiments the progesterone effect on estradiol nuclear receptors appeared to be of approximately 2-h duration, which coincided with the time period of progesterone nuclear receptor accumulation after a single injection of progesterone. During the period of progesterone effects on nuclear estrogen receptors, the ability of estrogens to induce progesterone receptors was impaired. Based on the above results, a model is proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger.

The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN'-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN'-dicyclohexylcarbodi-imide.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Circulating concentrations of thyroid hormones in pregnant camels (Camelus dromedarius )

Blood samples from 16 female camels were collected at monthly intervals commencing from 60 d post. breeding until the last month of gestation. Two camels failed to conceive and two had unnoticed abortions. The average gestation period was 398+/-13 and 372+/-11 in camels bearing male and female fetus, respectively, with an overall mean of 383+/-9 d. Sera were analyzed for thyroxine (T(4)) and triiodothyronine (T(3)) by radioimmunoassay. Mean T(4) and T(3) concentrations varied from 76 to 116 ng/ml and 0.73 to 1.32 ng/ml, respectively, during various stages of gestation. In general, the T(4) and T(3) levels were higher during early pregnancy, with lowest values in the tenth month. T(4):T(3) ratio showed minor, nonsignificant fluctuations. Age of dam of sex of fetus had no effect on hormone levels. Similarly, hormone levels were not affected by failure of conception or by abortion.     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

Binding properties of diethylcarbamazine

Diethylcarbamazine (DEC) reacted with liver cell plasma membrane of rodent hosts-cotton rat, albino rat and Mastomys natalensis exhibiting the presence of both saturable and unsaturable components. The presence of lectins or sugar derivatives did not affect the binding significantly. The drug showed similar binding pattern with serum but the saturation was reached at a much lower concentration of the ligand. Data obtained with a variety of macromolecules, particularly with the homopolymers of amino acids indicate that DEC does not require any specific constituent of the membrane for binding. The nonspecific nature of DEC binding does not provide any convincing clue for the accumulation of microfilariae specifically in the liver following the drug treatment.     

Sodium-gradient-driven, high-affinity, uphill transport of succinate in human placental brush-border membrane vesicles.

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.     

A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles

The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes.