Tissue dosimetry expansion and cross-validation of rat and mouse physiologically based pharmacokinetic models for trichloroethylene.

Trichloroethylene (TCE), a volatile liquid used as a degreasing agent, is a common environmental pollutant. In 2001, the EPA published a draft risk assessment for TCE that incorporates dosimetry predictions of physiologically based pharmacokinetic (PBPK) models. The current modeling effort represents an expansion and extensive tissue dosimetry validation of rodent PBPK models for TCE. The pharmacokinetics of TCE in male Sprague-Dawley (S-D) rats were characterized (1) during and after inhalation exposure to 50 or 500 ppm TCE, (2) following administration of 8 mg/kg TCE PO, and (3) following intra-arterial injection of 8 mg/kg TCE. Blood and tissues (including liver, kidney, fat, skeletal muscle, heart, spleen, gastrointestinal tract, and brain) were collected at selected time-points from 5 min up to 24 h post initial exposure. The fat compartment was modified to be diffusion-limited to predict the observed slow release of TCE from the fat. The addition of a deep liver compartment was necessary to accurately predict the slower hepatic clearance of TCE for all three exposure routes. Simulations of liver concentrations following gavage of male B6C3F1 mice with 300-2000 mg/kg TCE were also improved with the addition of a deep liver compartment. Liver predictions were calibrated and validated using a cross-validation technique novel to PBPK modeling. Splitting of compartments did not significantly affect predictions of TCE concentrations in the liver, fat, or venous blood. This model expansion and validation increases both the utility and our confidence in the current use of rodent TCE PBPK models in human health risk assessment.     

Prophylactic neuroprotective property of Centella asiatica against 3-nitropropionic acid induced oxidative stress and mitochondrial dysfunctions in brain regions of prepubertal mice

Despite the increasing popularity of Centella asiatica (a well known plant in ayurvedic medicine) globally, evidence demonstrating its protective efficacy against neurotoxicants in animal models is limited. 3-Nitropropionic acid (3-NPA), a fungal toxin is a well known neurotoxicant which induces selective striatal pathology similar to that seen in Huntington's disease. The present study aimed to understand the neuroprotective efficacy of a standardized aqueous extract of C. asiatica (CA) against 3-NPA-induced early oxidative stress and mitochondrial dysfunctions in striatum and other brain regions. We determined the extent of oxidative stress in cytosol and mitochondria of brain regions of male mice (4 wk old) given CA prophylaxis (5 mg/kg bw) for 10 days followed by 3-NPA administration (i.p., 75 mg/kg bw/d) on the last 2 days. The neurotoxicant elicited marked oxidative stress in the untreated mice as evidenced by elevated levels of malondialdehyde, ROS levels and hydroperoxides in the striatum (cytosol and mitochondria), while CA prophylaxis completely attenuated the 3-NPA-induced oxidative stress. 3-NPA also caused significant oxidative stress and protein oxidation in cytosol/mitochondria of other brain regions as well which were predominantly abolished by CA prophylaxis. Significant depletion of GSH levels, total thiols and perturbations in antioxidant enzymic defences in striatum and other brain regions discernible among 3-NPA administered mice were also protected with CA prophylaxis. Interestingly, CA prophylaxis offered varying degree of protection against 3-NPA-induced mitochondrial dysfunctions viz., reduction in the activity of succinic dehydrogenase, ETC enzymes and decreased mitochondrial viability. Collectively these findings clearly suggest that short-term oral intake of a standardized aqueous extract of CA confers marked resistance against the 3-NPA-induced oxidative stress and mitochondrial dysfunctions in brain. Although the precise mechanism/s underlying the prophylactic efficacy of CA merit further investigation, based on these findings, it is hypothesized that it may be wholly or in part related to the enhancement of GSH, thiols and antioxidant machinery in the brain regions of prepubertal mice.     

Assessment of in vivo mutagenic potency of ethylenediaminetetraacetic acid in albino mice.

Ethylenediaminetetraacetic acid (EDTA) disodium salt, a widely used metal chelator, was studied for its potency to induce bone marrow micronuclei, dominant lethal mutations and sperm-head abnormalities in albino mice. The acute oral LD₅₀ dose computed by probit regression was 30 mg/kg body weight in the strain used. Preliminary studies showed that oral administration of EDTA disodium salt at doses of 5, 10 and 15 mg/kg body weight/day on 5 consecutive days did not induce any obvious signs of toxicity. In the bone marrow micronucleus assay acute doses of EDTA disodium salt (5–20 mg/kg body weight) induced a dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes at a 24-hr sampling. However, administration at doses of 5, 10 and 15 mg/kg for 5 consecutive days did not produce any observable effect on either the testicular or epididymal weights and histology. No appreciable alterations were observed in the caudal sperm counts at any of the sampling intervals and there was no treatment-related increase in the incidence of sperm-head abnormalities. Furthermore, treatment of male mice with EDTA disodium salt (10 mg/kg body weight/day for 5 consecutive days) induced no increase in the incidence of post-implantation embryonic deaths, except for a marginal but statistically insignificant increase during wk 2 and 3 of mating.     

Assessment of in vivo mutagenic potency of ethylenediaminetetraacetic acid in albino mice

Ethylenediaminetetraacetic acid (EDTA) disodium salt, a widely used metal chelator, was studied for its potency to induce bone marrow micronuclei, dominant lethal mutations and sperm-head abnormalities in albino mice. The acute oral LD₅₀ dose computed by probit regression was 30 mg/kg body weight in the strain used. Preliminary studies showed that oral administration of EDTA disodium salt at doses of 5, 10 and 15 mg/kg body weight/day on 5 consecutive days did not induce any obvious signs of toxicity. In the bone marrow micronucleus assay acute doses of EDTA disodium salt (5–20 mg/kg body weight) induced a dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes at a 24-hr sampling. However, administration at doses of 5, 10 and 15 mg/kg for 5 consecutive days did not produce any observable effect on either the testicular or epididymal weights and histology. No appreciable alterations were observed in the caudal sperm counts at any of the sampling intervals and there was no treatment-related increase in the incidence of sperm-head abnormalities. Furthermore, treatment of male mice with EDTA disodium salt (10 mg/kg body weight/day for 5 consecutive days) induced no increase in the incidence of post-implantation embryonic deaths, except for a marginal but statistically insignificant increase during wk 2 and 3 of mating.     

Rational engineering of human cytochrome P450 2B6 for enhanced expression and stability: importance of a Leu264->Phe substitution

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed approximately 3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu(264)-->Phe substitution did not alter the K(s) for inhibitors or the substrate benzphetamine, the K(m) for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.     

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [¹²⁵I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [¹²⁵I]IAAP for the C-terminal half of the protein (C-site) by reducing the K_d from 20 to 6 nM without changing the labeling or affinity (K_d = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [¹²⁵I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [¹²⁵I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.     

Nutritional status and spontaneous locomotor activity in the rat

A considerable amount of energy may be saved by lowering the spontaneous locomotor activity when energy intakes are reduced. The results of the present study in rats undernourished for a period of 21 or 60 days and subsequently fed ad libitum diet did not show any differences in activity when compared to their respective control groups, either during the undernourished periods or well fed states. Although this would mean that the rats are not economising energy on activity, it is probable that these rats with lower body weights are contributing to energy saving mechanism by reducing the cost of activity per se since the cost of activity and body weight are directly related.     

Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal -Catalyzed Destabilization of a Therapeutic Monoclonal Antibody

Understanding the effect of metal chelators with respect to their ability to inhibit metal catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na₂EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation and fragmentation demonstrated that DTPA was equivalent to Na₂EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na2EDTA and DTPA exhibited the capacity to reduce protein degradation. However, sub-stoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb, but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na₂EDTA in biologic protein formulations.