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Poisoning with agricultural chemicals is a major threat for wildlife all over the world. We have developed and validated an analytical scheme aimed to the identification and quantification of a wide range of pesticides in fresh liver from wildlife specimens that are routinely delivered to the forensic laboratories for toxicological investigation. The proposed method is comprised of a general solid-liquid extraction followed by purification steps and three complementary liquid or gas chromatographic analyses with triple quadrupole mass spectrometry detection. The developed methodology allows for the determination of 117 highly toxic pesticides in a variety of samples from wildlife poisoning incidents. The validity of the method has been demonstrated in samples from 98 real cases submitted to our laboratory between 2010 and 2012. This method allowed the identification and quantification of poison in 78 of 94 fresh liver samples from wild animals and was successfully used for the identification of pesticides in 35 of 46 non-liver samples. Therefore, the extraction and cleanup method with minor modifications and the potency of triple quadrupole mass spectrometry allow this method to be used to simultaneously detect and quantify or semi-quantify a majority of the most toxic pesticides in a variety of complex and degraded matrices.

Some 17 alpha-alkylated androgens used as anabolic agents, such as stanozolol (ST) and danazol (DA), have specific effects on the liver that are not exerted by testosterone. This gives rise to the possibility that a steroid-binding protein, other than the androgen receptor, could modulate the intracellular actions of these agents. Male rat liver microsomes contain a homogeneous population of [3H]dexamethasone ([3H]DEX)-binding sites which we have denominated low affinity glucocorticoid-binding sites (LAGS). Because glucocorticoids, progestagens, and the synthetic estrogen ethynyl estradiol compete with [3H]DEX for binding to the LAGS, we aimed to study the possible interactions between androgens and the LAGS. To investigate whether several androgens had the capability of interacting with the LAGS, we performed competition experiments. The LAGS had no affinity for testosterone or methyltrienolone (R1881). However, some 17 alpha-alkylated androgens (DA (IC50, 116 nM) > ST >> fluoxymesterone > mestaline > methandriol >> methandrostenolone > methyltestosterone) were able to compete with [3H]DEX binding to liver microsomes. ST and DA were potent inhibitors of [3H]DEX binding to liver microsomes. They decreased both the affinity and the number of [3H]DEX-binding sites, increased the dissociation rate of [3H]DEX from the LAGS, and provoked a time- and dose-dependent inactivation of the [3H]DEX-binding site. These results strongly suggest that ST and DA exert a negative allosteric modulation on [3H]DEX binding to the LAGS. The in vivo administration of ST (but not other androgens) to male rats provoked a time- and dose-dependent decrease in the LAGS level. Full recovery of the LAGS concentration required at least 8 h and was blocked by protein synthesis inhibitors. Such results suggest that ST irreversibly inactivates the [3H]DEX-binding site in vivo as it does in vitro. Taken together, these observations are indicative of an irreversible interaction between some 17 alpha-alkylated androgens and the LAGS both in vitro and in vivo and suggest that ST may be an important pharmacological tool that can be used in the elucidation of the molecular structure of the LAGS. These results also mean that the LAGS are a steroid-binding entity able to distinguish between natural androgens and 17 alpha-alkylated testosterone derivatives used as anabolic agents.

GH participates in the regulation of the expression of several hepatic proteins, some of which are subject to multihormonal control. We have previously shown the participation of glucocorticoids and thyroid hormones in the regulation of the hepatic low affinity glucocorticoid-binding sites (LAGS). Here, we provide evidence that also implicates GH in the endocrine control of the LAGS through the use of several animal models, all of them having a very low or undetectable plasma GH level: the hypothyroid (TX), the hypophysectomized, and the GH-deficient Lewis-derived dwarf rat. In dwarf rats, the level of LAGS was only 35% of that found in normal Lewis rats. Treatment of these rats with human (h) GH significantly increased the LAGS level in a dose-response manner. In TX rats, hGH treatment provoked a significant increase in the LAGS level (from 0.9 +/- 0.2 to 7.2 +/- 0.8 pmol/mg protein), so that it represented about 65% of the level found in intact animals. In both hypothyroid-adrenalectomized and hypophysectomized rats, the isolated effect of hGH was not as pronounced as in TX or dwarf rats; however, a potentiation of the effect of hGH was observed when this hormone was injected together with corticosterone acetate. On the other hand, when hGH, T3, and corticosterone acetate were given in combination to hypophysectomized rats, hGH and T3 behaved as agonists of the LAGS induction at T3 doses lower than or equal to 0.1 microgram/100 g BW and as antagonists at T3 doses higher than this. When T4 was used instead of T3, this hormone was capable of potentiating the effect of hGH at doses lower than or equal to 1.5 micrograms/100 g BW. From these results we conclude that 1) GH as well as thyroid and glucocorticoid hormones participate in the endocrine regulation of the LAGS; and 2) under physiological conditions, it is conceivable that GH, thyroid hormones, and glucocorticoids act synergistically in the endocrine regulation of the LAGS.