Changes in circulating concentrations of testosterone and estrone sulfate after human chorionic gonadotropin administration and subsequent to castration of 2-year-old stallions.

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

Risk assessment of substances that are both genotoxic and carcinogenic report of an International Conference organized by EFSA and WHO with support of ILSI Europe.

The European Food Safety Authority (EFSA) and the World Health Organization (WHO), with the support of the International Life Sciences Institute, European Branch (ILSI Europe), organized an international conference on 16-18 November 2005 to discuss how regulatory and advisory bodies evaluate the potential risks of the presence in food of substances that are both genotoxic and carcinogenic. The objectives of the conference were to discuss the possible approaches for risk assessment of such substances, how the approaches may be interpreted and whether they meet the needs of risk managers. ALARA (as low as reasonably achievable) provides advice based solely on hazard identification and does not take into account either potency or human exposure. The use of quantitative low-dose extrapolation of dose-response data from an animal bioassay raises numerous scientific uncertainties related to the selection of mathematical models and extrapolation down to levels of human exposure. There was consensus that the margin of exposure (MOE) was the preferred approach because it is based on the available animal dose-response data, without extrapolation, and on human exposures. The MOE can be used for prioritisation of risk management actions but the conference recognised that it is difficult to interpret it in terms of health risk.

A comparison of the ability of beta-galactosidase and horseradish peroxidase enzyme-antibody conjugates to detect specific antibodies.

The ability of two different enzyme-antibody conjugates to detect specific antibodies has been compared. beta-Galactosidase was conjugated to antibodies raised against rabbit Fc fragments using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Horseradish peroxidase (HRP) was conjugated to part of the same batch of antibodies using the periodate method. The beta-galactosidase and HRP labels enabled detection of approximately 8 fmoles and 4 fmoles respectively of human growth hormone (HGH) antibodies, when their enzyme activities were measured spectrophotometrically. The detection limit of the beta-galactosidase label was increased 4-fold when a fluorimetric detection system was employed.

The influence of antigen properties on the conditions required to elute antibodies from immunoadsorbents.

Immunoadsorbents were used to purify a number of antibodies. Using pH 2.0 acid conditions alone it was possible to elute antibodies raised against human growth hormone (HGH) and Fc fragments from such immunoadsorbents with 50% or better recovery of antibody activity. However, to elute antibodies raised against triiodothyronine and cortisol required 6 M guanidine HCl, pH 2.0. The avidities of the purified antibodies were similar to those of the non-purified antibodies. The purified antibodies were stable in solution at 4 degrees C for at least six months.

Rapid absorption from the urinary bladder of a series of n-alkyl carbamates: a route for the recirculation of drugs.

1 The rate of loss of a series of n-alkyl carbamates from the lumen of the urinary bladders of female rats has been studied. 2 The rate of loss obeys first order kinetics and was not affected by water flux across the bladder wall nor by binding of the carbamates to it. 3 The rate of loss of octyl carbamate was reduced by about 76% by the presence of 5% Tween 80. Histological evidence indicates that this may be due to the formation of a thin luminal lining which may be adsorbed Tween 80 or mucopolysaccharide material. 4 The absorption rate of the carbamates was limited by their hydrophilicity but reached a plateau for the more lipophilic homologues with a half life for loss of approximately 10 min. 5 The implications of these results with regard to the recirculation of unmetabolized drugs and hydrolysed conjugates of drugs, the systemic absorption of intravesically applied cancer chemotherapeutic agents and bladder wall permeability to carcinogens are discussed.

The relationship between the binding of 2-n-alkylbenzimidazoles to rat hepatic microsomal cytochrome P-450 and the inhibition of monooxygenation.

The binding of a homologous series of 2-n-alkylbenzimidazoles to rat hepatic microsomal cytochrome P-450 has been examined. Type I, Type RI and mixed Type I/RI spectra were observed with control, phenobarbitone or 20-methylcholanthrene-induced microsomal preparations. In general short chain (C1-C4) substituted compounds elicited Type RI spectra, whereas C5-C9 substituted benzimidazoles gave rise to Type RI/I or Type I spectra. The type of binding spectrum observed was dependent upon the substrate concentration, the source of microsomes and the length of the substituent alkyl chain. As the lipophilic character of the substituent was increased a corresponding increase in Type I nature was noted. However, an optimal chain length of C7-C8 carbon atoms was observed for Type I binding; compounds with longer side chains showed a decreased affinity for the Type I site. The apparent spectral binding constants (Ks values) for the Type I site (but not the Type RI site) were closely associated with the Ki and I50 values for the inhibition of cytochrome P-450-dependent monooxygenation. From their inhibition properties it seems that even the short chain (C1-C4) substituted benzimidazoles also bind toi the Type I site and thus compete for the substrate binding site of cytochrome P-450.

Guinea-pig intestinal sulphotransferases: an investigation using the cytosol fraction.

A rat liver cytosol preparation fortified with a PAPS regenerating system has been used to study enzymically mediated sulpha conjugation of several phenols. In agreement with previous findings with isolated intestinal epithelial cells and hepatocytes from rats 2-hydroxybiphenyl was poorly sulphated compared with 4-hydroxybiphenyl and 7-hydroxycoumarin. The results are attributed to the involvement of different sulphotransferases in the conjugation of these substrates. Examination of the effects of changing pH and substrate concentration indicated that at least two sulphotransferases are probably involved in the sulphation of 2-hydroxybiphenyl and four sulphotransferases participate in the sulphation of 4-hydroxybiphenyl.

Intestinal microsomal drug metabolism. A comparison of rat and guinea-pig enzymes, and of rat crypt and villous tip cell enzymes.

A comparison was made of the properties of microsomes prepared from the small intestines of guinea pigs and rats. The NADPH2 cytochrome c reductase activity and cytochrome b5 and cytochrome P-450 content in rat microsomes was 42, 47 and 64% of that in the guinea pig, ethoxycoumarin deethylase activity was comparable, while arylesterase activity was twice as active in rats as guinea pigs. Investigation of the distribution of these and other parameters in rat intestinal epithelia revealed a preferential location of cytochrome P-450 in the villous tip while other parameters showed a more similar distribution between microsomes prepared from the villous tip and crypt.

Mechanistic studies on the activation of biphenyl 2-hydroxylation by glucocorticoids.

In order to establish the mechanism by which the selective activation of biphenyl 2-hydroxylation by betamethasone occurs the effect of modifying possible critical factors in the hydroxylation process has been examined. Activation of biphenyl 2-hydroxylation by betamethasone was found in detergent-solubilized rat liver microsomes indicating that intact microsomal membranes are probably not necessary for the activation. Betamethasone had no effect on the spectrally apparent binding of biphenyl or of other type I, type II or reverse type I model substrates. The activation process did not appear to be greatly influenced by changing the ratio of cytochrome P-450 reductase to cytochrome P-450 nor by changing the amount of NADPH. Addition of NADH increased the extent of activation suggesting that betamethasone facilitates transference of the second electron to cytochrome P-450. However, betamethasone also stimulated cumene hydroperoxide supported biphenyl 2-hydroxylation; therefore a step subsequent to cytochrome P-450 reduction is also involved in the activation. Activation did not correlate with increased uncoupling of an active oxygen-cytochrome P-450 complex to form hydrogen peroxide.

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats.

Betamethasone and alpha-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas alpha-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas alpha-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes female rats betamethasone activated whereas alpha-naphthoflavone had no effect. Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one 'latent' aromatic hydrocarbon hydroxylase in rodents.

Effects of mono (2-ethylhexyl) phthalate and its straight chain analogues mono-n-hexylphthalate and mono-n-octyl phthalate on lipid metabolism in isolated hepatocytes.

In cultured hepatocytes, as in vivo, mono-2-ethylhexyl phthalate (MEHP) and its straight chain analogues mono-n-hexyl phthalate (MnHP) and mono-n-octyl phthalate (MnOP) each cause accumulation of lipid but only MEHP produces significant induction of peroxisomal fatty acid oxidizing enzymes. To elucidate the mechanisms underlying this lipid accumulation we investigated the effects of these phthalates and the drug clofibric acid on fatty acid metabolism in suspensions of isolated hepatocytes. The effects were found to be markedly dependent on the nutritional state of the animals from which the hepatocytes were isolated. In hepatocytes isolated from animals fasted overnight, or animals fed ab libitum but killed at approximately 2.30 p.m., MEHP, MnHP, MnOP and clofibric acid each caused a marked rapid stimulation of fatty acid oxidation and the synthesis of triglycerides in hepatocytes when incubated in Hanks saline. Export of very low density lipoprotein (VLDL) from the cells was either unchanged or somewhat reduced. In contrast, in hepatocytes isolated from rats fed ad libitum but killed at approximately 9.30 a.m. MEHP and clofibric acid did not alter fatty acid oxidation or triglyceride synthesis, while MnOP and MnHP increased triglyceride synthesis but decreased fatty acid oxidation. The effects of fasting were largely abolished by incubations of the cells in a complete tissue culture medium (Liebowitz L-15). The results suggest that MEHP and its straight chain analogues can, either as the free acid or the CoA ester, mimic the action of fatty acids in the allosteric regulation of fatty acid metabolism.

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes.

The binding constants of an homologous series of n-alkyl (C2-C10) carbamates (see formula in text) to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/Ks) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. Th major metabolites of C4-C8 carbamates were unconjugated omega-1 oxidation products. Below hexyl carbamate only the omega-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the omega-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin by rat and hairless mouse skin strips.

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was studied in rat and hairless mouse skin strips. These preparations supported de-ethylation, sulphation and glucuronidation reactions. The de-ethylation reaction was inducible in both species by pretreatment with either 5,6-benzoflavone or 3-methylcholanthrene. The hairless mouse strips exhibited a greater basal de-ethylase activity than rat strips, although the latter was the more responsive to inducers. Accompanying the increase in de-ethylation activity was a change in the pattern of metabolites, with a large increase in the percentage of the unconjugated metabolite. When 7-hydroxycoumarin was employed as the primary substrate the glucuronide was the major metabolite formed by strips from both species. The glucuronidation and sulphation activities were unchanged by 3-methylcholanthrene pretreatment.

Cytochrome P-450 dependent deethylase activity in rat and hairless mouse skin microsomes.

Microsomal fractions were prepared from rat and hairless mouse skin. The method of preparation was validated by studying the distribution of succinate dehydrogenase, acid phosphatase and UDP-glucuronosyltransferase. Induction of oxidative deethylation activities by 5,6-benzoflavone and 3-methylcholanthrene was investigated. Preparations from hairless mouse skin exhibited higher basal activities but the enzymes were less responsive than those of rat skin to inducers. Species differences were observed in the extent of induction between topical and i.p. administration of 5,6-benzoflavone, the former route being more effective in the hairless mouse and the latter route most effective in the rat. Generally oxidative deethylation activity increased linearly with protein concn up to 2-3 mg protein/ml. The only exception was rat skin microsomes prepared from animals pretreated with 5,6-benzoflavone, where linearity was observed only to 0.75 mg protein/ml above which oxidative deethylation activity decreased with increasing protein concn. The inhibition of 7-ethoxycoumarin deethylase by various compounds was investigated; the activity in hairless mouse skin exhibited a greater sensitivity to water-soluble solvents than that in rat skin microsomes. Both hairless mouse and rat skin 7-ethoxycoumarin deethylase were sensitive to inhibition by 5,6-benzoflavone, 7,8-benzoflavone and metyrapone.

Chain length dependency of fatty acid and carbamate binding to serum albumin.

The binding interactions of bovine serum albumin (BSA) with the unbranched fatty acids (FA) pentanoate (five-carbon chain length: C5) up to nonanoate (C9), and the carbamates n-methyl carbamate (equivalent to C3) up to n-hexyl carbamate (equivalent to C8) were examined using an ultrafiltration technique. A single, high-affinity site was observed for each of the FA, with an increasing number of secondary sites with increasing chain length. From binding affinity and competition data, there appear to be distinct albumin sites for the short-chain (less than or equal to C7) and the medium-chain (greater than or equal to C8) FA. Published data suggest that the medium-chain FA site is one of the major drug-binding sites on human serum albumin (HSA) or BSA, the indole/benzodiazepine site. Competition between the FA and warfarin for BSA or HSA binding was studied by ultrafiltration and fluorescence methods and suggests that the short-chain FA site may lie in the same region as a second major drug-binding site, the large warfarin-binding area. Thermodynamic parameters of the FA-BSA interactions are suggestive of primary binding being a combination of electrostatic and hydrophobic binding and secondary binding being purely hydrophobic in nature. Carbamate interactions with BSA show several primary sites and also suggest a disparity between the binding of ligands of less than or equal to 7 and greater than or equal to 8 in total length, but there was no evidence of competition between FA and carbamates. A model is proposed to explain these observations, which includes the suggestion that several classes of hydrophobic binding areas exist, each of which is specific for ligands of a restricted range of chain lengths.

Effects of phthalic acid esters on the liver and thyroid.

The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P-450 isoenzyme(s) catalyzing omega oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fail to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid. The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.

The metabolism of 7-ethoxycoumarin in primary maintenance cultures of adult rat hepatocytes.

7-Ethoxycoumarin is metabolized to 7-hydroxycoumarin in short-term (1-4 days) maintenance cultures of adult rat hepatocytes. The 7-hydroxycoumarin is predominantly found as the sulphate and glucuronic acid conjugates. This pattern of metabolism is very similar to that observed with freshly-isolated rat hepatocytes and suggests that the culture system may be of value in studying the metabolism of novel chemicals designed for human therapeutic use. The specific activity of microsomal monooxygenase activity falls by 50-60% during 4 days in culture. This is not reflected by the sulphate and glucuronic acid conjugation pathways which are retained at normal levels throughout the entire 4-day culture period.

The effect of some glucocorticoids on hepatic microsomal hydroxylation in the rat and hamster.

The effect of some glucocorticoids on hepatic microsomal biphenyl and aniline hydroxylations were investigated in rat and hamster. Steroids were either added to the standard incubation mixture or given as a single dose intraperitoneally before preparation of the liver microsomes for enzyme determination. The addition of steroids in vitro enhanced biphenyl-2-hydroxylation activity in hepatic microsomes of rat but not of hamster and the most pronounced effect was obtained with betamethasone. A similar species difference in the effects of betamethasone on this enzyme was also observed after administration of a single dose of the steroid in vivo.

Integrated risk assessment and endocrine disrupters.

1. The procedures currently employed for risk assessment are unlikely to be sustainable in the future for a variety of reasons. A number of actions are needed to remedy the situation and the most important of these actions are: * to improve access to existing data; * to introduce a prioritisation system based on exposure assessment; * to co-ordinate and harmonise approaches of different organisations involved in risk assessment. 2. Integration of information and methodologies between human health risk assessment and ecological risk assessment (integrated risk assessment) is advocated in this paper as one of the most important steps towards a holistic and effective way of conducting a risk assessment. 3. A framework is proposed for identification of agents for which an integrated risk assessment would be of particular value. Close collaboration across disciplines and across countries is necessary for the potential of integrated risk assessment to be realised in practice. The practicality of applying integrated risk assessment to endocrine disrupting agents is presently being investigated in an EU funded multi-laboratory collaborative study (CREDO).

The effects of dimethylnitrosamine and allyl alcohol on primary maintenance cultures of adult rabbit hepatocytes.

Cultures of adult rabbit hepatocytes have been used to study the early toxic effects of 2 model hepatotoxins, dimethylnitrosamine and allyl alcohol. Leakage of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase into the cell culture medium was a sensitive indicator of plasma membrane damage by these compounds and a dose-response relationship was observed. By contrast, gamma-glutamyltranspeptidase and alkaline phosphatase were insensitive markers. The effects of dimethylnitrosamine were slower to develop. Dimethylnitrosamine also produced a dose-related inhibition of protein synthesis after 4 h, a decrease in NADPH diaphorase and an increase in non-specific esterase after 20 h. Dimethylnitrosamine, unlike allyl alcohol, caused extensive disruption of ribosome association with the endoplasmic reticulum.