Effects of parenteral di-(2-ethylhexyl)phthalate (DEHP) on gonadal biochemistry, pathology, and reproductive performance of mice.

Male and female mice were treated subcutaneously (sc) with 1-100 ml/kg of di-(2-ethylhexyl) phthalate (DEHP) on d 1, 5, and 10 of the experiment and evaluated at d 21 for reproductive performance, selected biochemical parameters of the gonads, and histological alterations of the gonads. In both male and female treated mice there was a reduction in incidence of pregnancy. There were biochemical suggestions of reduced anabolic activity in the gonads (as reflected by decreased ATPase activity and of RNA, DNA, and protein content), and of increased catabolic activity in the gonads (as reflected by an increase in lysosomal enzyme activity and histological damage). Testicular, but not ovarian, weight was reduced in treated animals. Of the other parameters examined, the ovaries exhibited histological injury at lower doses of DEHP than the testes, but unlike testes, there was not a significant dose-related increase in histopathology. Biochemical changes were dose-related, for the most part, in both ovaries and testes, with the changes being more pronounced in testes. In general, reduced fertility appeared to be the most sensitive indicator for gonadotoxicity from DEHP, followed by biochemical changes and histological evidence of injury to the gonads.

Effects of phthalic acid esters on drug metabolizing enzymes of rat liver.

Di(2-ethylhexyl)phthalate (DEHP) inhibited UDP-glucuronyltransferase activity of rat liver in vitro and in vivo. Diethyl phthalate and dimethoxyethyl phthalate also inhibited this enzyme in vitro. On the other hand, DEHP did not inhibit the activity of the cytosolic enzyme N-acetyltransferase; it also did not alter the levels of rat liver microsomal cytochrome P-450 in vitro. It is suggested that DEHP may alter the composition of microsomal phospholipids.

Antifertility and mutagenic effects in mice from parenteral administration of di-2-ethylhexyl phthalate (DEHP).

The subcutaneous administration of 1-10 mg of undiluted di-(2-ethylhexyl)phthalate di-(2-ethylhexyl)phthalate (DEHP) to adult male ICR mice on d 1, 5, and 10 was followed by mating, one to one, with untreated adult virgin females. A single mating at d 21 resulted in a reduction in the incidence of pregnancies in the DEHP-treated groups. On the other hand, repeated matings with fresh females starting on d 2, 6, 11, 16, and 21, and at weekly intervals through 8 wk, revealed no perceptible effect of DEHP on the incidence of pregnancy. Examination of surgically exposed uteri and ovaries of pregnant females on d 13 of gestation revealed an increase in the incidence of preimplantation losses and early fetal deaths in the DEHP-treated groups; consequently, there were fewer viable fetuses per pregnancy. Mutagenic indices for DEHP, calculated as percent ratios of (1) preimplantation losses/implantations per pregnancy and (2) early fetal deaths/implantations per pregnancy, suggested a dominant lethal mutation effect in the treated mice. These effects tend to be more pronounced on the postmiotic stage of germ-cell development.

Developmental changes in the conversion rates of di(2-ethylhexyl) phthalate to monoethylhexyl phthalate in rats.

The activities of liver, lung, and kidney of rats of various age groups and that of placenta in hydrolyzing di(2-ethylhexyl) phthalate to mono(2-ethylhexyl) phthalate have been measured. Male and female rats of 45 d of age, neonatal rats within 12 h of parturition, and fetuses and placenta on d 19 of gestation were used. The liver was most active in all age groups; however, the lung and the kidney also had considerable activity. The tissues of the fetuses and the neonate had significant activity. The Km values of the enzyme were 4 mM in the neonatal liver and 5.9 mM in the adult liver.

Mutagenicity evaluation of phthalic acid esters and metabolites in Salmonella typhimurium cultures.

The mutagenic potential of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEPH), as well as metabolites of DEHP--i.e., mono-2-ethylhexyl phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA)--were tested in Salmonella typhimurium cultures using the Ames test procedure. The compounds were tested on strains TA98, TA100, TA1535, TA1537, TA1538, and TA2637 for base-pair substitution or frameshift-type mutations. Spot tests yielded negative responses for all compounds with the strains tested. Each compound was tested for a dose-effect relationship in the TA98, TA100, TA1535, and TA1538 systems. DEP and DBP exhibited a mildly positive response in both TA100 and TA1535 cultures, and DMP showed a similar response in TA1535. Normalization of the data for cytotoxicity of DMP suggests TA100 has a mildly positive effect. The higher doses of these compounds exhibited some cytotoxic effects. The mutagenic effects were apparently abolished by the addition of S9 fraction in TA100 and TA1535 cultures, while no effect, other than cytotoxicity, was observed in the TA98 and TA1538 systems. DEHP, MEHP, 2-EH, and PA exhibited no mutagenicity in any of the strains of Salmonella typhimurium tested, with or without S9 metabolic activation. MEHP and 2-EH, however, exhibited a moderate cytotoxic effect in most cultures.

Acrylate and methacrylate esters: relationship of hemolytic activity and in vivo toxicity.

Quantitative hemolysis assays of acrylate and methacrylate esters provided estimates of the intrinsic hemolytic activity (Hi, the slope of the concentration-response curve) and the concentrations effecting 5% (H5) and 50% (H50) hemolysis. The dependence of hemolytic activity and LD50 (mice) on physical properties (lipophilicity, molar refraction, and molecular volume) of the esters was determined by multiple regression analysis. The observed correlations were: Hi, R2 = 0.94; H5, R2 = 0.95; H50, R2 = 0.94; and LD50, R2 (all compounds) = 0.80, R2 (all compounds less the methyl esters) = 0.94. The difference of the methyl esters was associated with the smaller steric volume of the methyl ester substituent and the presence (methacrylates) or absence (acrylates) of the branched methyl group. Associative steric contributions of the branched methyl group and the ester substituents were probably responsible for greater variability in the methyacrylate series. The results were consistent with the conclusion that the mechanism of the action of the esters is membrane mediated and relatively nonspecific and that in vivo biotransformation was not a significant factor. Also, long-term toxic liability of the esters may be more closely related to intrinsic toxicity than acute toxicity.

Impaired metabolism of di(2-ethylhexyl) phthalate (DEHP) in old rats--an in vitro study.

Liver, kidney, and lung homogenates from 45- and 630-d-old rats were incubated with 0.5 muCi of 14C-labeled di(2-ethylhexyl) phthalate (DEHP) for 40 min at 37 degrees C. Radiochromatogram scans of the ether extracts of the incubated mixtures showed the presence of a peak corresponding to the hydrolytic product monoethylhexyl phthalate (MEHP), in addition to the parent DEHP. Preparations from old animals revealed another radioactive zone along with MEHP. A dramatic reduction in the formation of MEHP was observed only with homogenates of livers from the old animals. It is shown that this difference is probably attributable to differences in Km values of the enzymes from adult and old rats respectively. Protein content in the three tissues did not differ between young and old animals. Formation of MEHP is a major step in the metabolic pathway of the plasticizer DEHP. Impairment of this conversion could possibly alter the rate of its excretion and hence its toxicologic significance.

The influence of urea and cell-surface protein on the behavior of nontumorigenic and chemically transformed cells.

Alterations in cell-cell interactions induced by urea and urea-isolated cell-surface protein (CSP) were examined using the nontumorigenic mouse fibroblast 10T1/2 cell line and the malignantly transformed MCA daughter cell line as a model system. Both cell lines were exposed to urea and CSP, and contact inhibition was quantitated based on nuclear overlaps. The Abercrombie Overlap index was found to be dependent on cell density, and a new method of overlap analysis was developed based on the regression of the number of overlaps per microscope field on the number of cells per field. Urea had a differential effect on both protein and deoxyribonucleic acid synthesis and a differential effect on the overlap regression in the two cell lines. Urea increased the regression coefficient in the normal (10T1/2) cell line while decreasing it in the transformed (MCA) cell line to approximately equal levels of overlap. Added CSP had no effects on overlaps in the MCA line but increased overlaps in the 10T1/2 line to the level of the MCA control, suggesting that the CSP interactions were more specific than the urea interactions. Trypsin-derived cell-surface glycopeptide profiles of the two cell lines showed a difference consistent with previously reported differences between normal and transformed cell lines. However, the glycopeptide profile of the urea-isolated CSP from the normal 10T1/2 cell line and the trypsin-derived glycopeptides of the transformed MCA cell line were not significantly different in the high-molecular-weight region, suggesting that the relative abundance of CSP may be higher in the MCA line, and the fact that the addition of CSP to the 10T1/2 line increased overlap tendency to the level of the MCA line suggested that CSP is an important factor in the modulation of overlap tendency. Urea and CSP may exert their effects on overlap tendency by affecting the integrity or order of the cell-surface components. The increased overlap tendency of the 10T1/2 line in the presence of CSP was not associated with increased cell density. Density dependence of cell growth was apparently not directly related to density dependence of overlap tendency. Many xenobiotics have surface-active properties and are known to inhibit protein synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

A new substrate for the measurement of N-acetyltransferase activity.

A sensitive colorimetric method for the measurement of N-acetyltransferase (NAT) is described. It is based on the high rate of acetylation of 2-(p-aminobenzamido)pyridine by the liver enzyme and the lack of it by the blood NAT. A linear relationship was found between enzyme concentration and reaction rate. The reaction rate was also proportional to the substrate concentration. Inhibition of the reaction was observed at high substrate concentrations. The NAT levels in the liver and kidney of rat, rabbit, mouse and man were measured using this procedure. The tissues of dog failed to acetylate this substrate. The method is applicable to kinetic studies such as the analysis of inhibition reactions with o-phenanthroline and p-chloromercuribenzoate.

Antifertility effects and dominant lethal assays for mutagenic effects of DEHP.

Results of past animal studies have indicated the antifertility effects of phthalate esters and additional studies have suggested the potential mutagenic effects at very high doses. A toxicity study in mice has also been conducted in which chronic LD50 values were calculated for a group of phthalate esters. For DEHP, for example, the acute LD50 was 38.35 ml/kg, but after 10 weeks the value fell to 1.37 ml/kg, suggesting that the ester was producing a cumulative toxicity agent even at lower doses in regard to antifertility and mutagenic effects in mice. Preliminary results indicated that antifertility effects occurred with as little as three subcutaneous doses of 1 ml/kg each. Calculations of mutagenic index revealed a dose-dependent effect but no stoichiometric relationship was established.

Clinical toxicology consultation by pharmacists.

The functions of pharmacists in a clinical toxicology consultation service are described. Pharmacists provided three basic services in conjunction with poisonings treated by the emergency department of a children's hospital: (1) assisted with obtaining the history and assessment of the toxicologic proglems, (2) recommended a plan for rational management, and (3) discussed poison prevention with the parents of the victims. Pharmacists were consulted for 189 poisoning cases over a six-month period; 80% of the cases were attended at the bedside and the remainder were monitored by telephone. Drugs were involved in 58% of the patient exposures. Median time for the pharmacist to reach the emergency room after the patient's arrival was 5 to 10 minutes. Physicians and nurses rated the pharmacists' contributions favorably. These results suggest that pharmacists can play an important role in clinical toxicology.