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This study was conducted to investigate if biotransformation of N,N,N-triethylene-thiophosphoramide (thio-TEPA) by liver slice incubations reflects the established gender pattern for rat and mouse. Liver slices from rat and mice of both genders were incubated with start concentrations of thio-TEPA of 5.2, 26, 52 and 104 microM for up to 240 min. Male rat liver slices eliminated thio-TEPA faster and formed more TEPA than female liver slices at any concentration. No gender difference was found for the elimination of thio-TEPA in mice, however, the female liver slices formed less TEPA than the male ones. Apparently female rat liver slices formed less TEPA than female mice liver slices. It is concluded that the liver slice incubation system in a robust manner reflects gender differences in rat drug biotransformation with special reference to thio-TEPA. It is also confirmed that these aspects of gender are less pronounced in the examined mouse species than in rats.

Surveys of Norwegian industrial occupational atmosphere between 1983 to 1996, have identified the 12 most frequent occurring binary combinations of volatile organic chemicals. These combinations were tested in vitro for mutual inhibition or enhancement of metabolism by the head space vial equilibration technique with liver S9 obtained from in vivo untreated or pretreated (with the binary mixture) rats. The in vitro system responded to in vivo pretreatment by increasing the metabolic rate of several potentially toxic organic chemicals such as toluene, xylene, styrene, and dichloromethane. In untreated liver S9, the metabolism of several of the tested binary pairs was inhibited when coexposed in vitro to their most prevalent follower as shown for instance for ethanol (with ethyl acetate), dichloromethane (with styrene) and mutually between toluene and xylene. This inhibitory effect disappeared, however, for several of the solvents when combined with the in vivo induced liver S9, a situation which may be the most relevant for occupational exposure. It is concluded that several metabolic interactions occur between low-molecular weight volatile chemicals found in occupational air. These are both inductive and inhibitory in nature and a further mechanistic evaluation including a higher number of differentiated dosage levels, must be performed before a possible health hazard can be confirmed or rejected for the investigated combinations.

The liver S9 head space vial equilibration technique is an in vitro alternative that holds promises for a satisfactory in vivo extrapolation of liver metabolism of volatile organic chemicals. The aim of this study was to investigate the suitability of this methodology for the extrapolation of in vitro metabolic data from rodent to man by allometry with the two highly metabolized organic solvents toluene and n-hexane as model substances. The calculated hepatic clearance of toluene in man from rodent liver S9 in this study was equal to the reported total body clearance of toluene in man, suggesting insignificant extrahepatic clearance of toluene in humans. The calculated hepatic clearance of n-hexane was less than the reported values of total body clearance of n-hexane in man, indicating an about 80% extrahepatic clearance of n-hexane in humans. Both results are in line with our present knowledge of the metabolism of the two organic solvents in man. Allometric scaling from rodent liver S9 head space incubations to in vivo metabolism of toluene and n-hexane in man thus seems promising and could be a method of choice for scaling of organic solvent metabolism in general.

A head space liver S9 vial equilibration technique for qualitative and quantitative metabolism of volatile organic solvents in vitro is described and investigated for optimal experimental conditions with toluene and n-hexane as model substrates. The method was also used for studying the metabolic interaction between acetone and styrene. NADP+ was a critical cofactor in the system as concentrations above 2.5 mM resulted in inhibition of metabolism. Induction of cytochrome P450 by phenobarbital did not alter the observed cofactor dependency, and addition of vehicles such as rat blood did not enhance metabolism. Acetone-induced liver S9 increased the primary oxidation of styrene and also the formation of styrene oxide, but not significantly that of mandelic acid. Acetone as such had no effect on the metabolic elimination of styrene, indicating that the interaction is mainly on the level of enzyme induction occurring after long-term exposure to acetone. As earlier reports on styrene metabolism in vivo has not been able to detect styrene oxide in toxicokinetic studies with a sufficient degree of accuracy, the head space liver S9 vial equilibration technique may offer a fast and sensitive tool for screening of metabolic interactions between organic solvents when optimized for metabolic activity.

Methyl ethyl ketone pretreatment induced rat liver cytochrome P450 and increased significantly the in vitro metabolism of n-hexane and the formation of 2,5-hexanedione in rat liver S9. No significant changes were, however, found in the levels of the intermediate metabolites 2-hexanol, 2,5-hexanediol or methyl n-butyl ketone. Methyl ethyl ketone added in vitro to untreated (non-induced) liver S9 inhibited in a non-competitive pattern the metabolism of n-hexane and decreased significantly and in a dose-dependent way the levels of methyl n-butyl ketone and 2,5-hexanedione. When methyl ethyl ketone and n-hexane were added in vitro to in vivo methyl ethyl ketone pretreated (induced liver S9, the significant increase in the formation of 2,5-hexanedione was maintained, an increase which was only to a minor extent influenced by the in vitro addition of methyl ethyl ketone. These findings are in agreement with an in vivo induction by methyl ethyl ketone of key enzyme(s) in a generally minor metabolic pathway for the conversion of n-hexane to 2,5-hexanedione in rat liver, a pathway which is not influenced by the presence of methyl ethyl ketone itself. The results obtained in this study indicate that the head space equilibration technique is well suited for screening studies of metabolic interactions between organic solvents.

In vitro systems of high biological organization, e.g. containing intact hepatocytes, have been considered as more reliable for metabolic studies and in vivo predictions of toxicokinetics than subcellular systems. For this reason, the kinetics and metabolism of low-molecular-weight volatile chemicals, i.e. head space elimination of substrate and formation of metabolites, were compared in liver S9 and in liver slices. Two substrates, toluene and n-hexane, were used as they represent differences in metabolic pathways and physical-chemical properties. The two systems responded similarly to diethyldithiocarbamate inhibition and acetone inhibition/induction of cytochrome P-450 mediated metabolism. In contrast to liver S9, liver slices did not respond adequately to phenobarbital induction raising the question of substrate availability for P-450 enzymes in liver slices. Liver slices appeared to be superior to liver S9 when specific metabolic pathways involving phase II enzymes were investigated. However, liver S9 seemed to be at least as good as liver slices for estimation of total metabolic rate constants (Km, Vmax) as a basis for in vivo predictions. As the head space liver S9 system is faster and easier to operate than liver slices, it is a promising screening tool for the metabolism of volatile compounds and metabolic interactions.

Previous studies (Haug & Gautvik 1985) have demonstrated specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a clonal (GH3) strain of rat pituitary tumour cells. It was discovered that 1,25(OH)2D3 affected the production of prolactin and growth hormone in these cells in a calcium dependent manner. These findings were the basis for a hypothesis that vitamin D3 could be involved in the regulation of pituitary hormones in vivo. To further investigate this contention, female rats were given subcutaneous injections of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25(OH)D3) or 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) three times a week for up to 12 weeks. Blood samples were withdrawn after 28, 56 and 84 days of treatment and analysed for vitamin D3 metabolites, prolactin and growth hormone, and serum ionized (free) and total calcium (Ca). Between treatment group comparisons of serum prolactin and growth hormone levels did not show significant vitamin D3 induced alterations. However, correlation matrix analyses on all variables revealed that serum level of growth hormone was significantly (P < 0.05) and inversely related to corresponding total Ca. Prolactin, on the other hand, may be subject to a complex regulation by 1,25(OH)2D3 and free Ca2+.