Evaluation of Therapeutics for Advanced-Stage Heart Failure and Other Severely-Debilitating or Life-Threatening Diseases.

Severely-debilitating or life-threatening (SDLT) diseases include conditions in which life expectancy is short or quality of life is greatly diminished despite available therapies. As such, the medical context for SDLT diseases is comparable to advanced cancer and the benefit vs. risk assessment and development of SDLT disease therapeutics should be similar to that of advanced cancer therapeutics. A streamlined development approach would allow patients with SDLT conditions earlier access to therapeutics and increase the speed of progression through development. In addition, this will likely increase the SDLT disease therapeutic pipeline, directly benefiting patients and reducing the economic and societal burden of SDLT conditions. Using advanced-stage heart failure (HF) as an example that illustrates the concepts applicable to other SDLT indications, this article proposes a streamlined development paradigm for SDLT disease therapeutics and recommends development of aligned global regulatory guidance.

Combinatorial solar cell libraries for the investigation of different metal back contacts for TiO2-Cu2O hetero-junction solar cells.

Here we present a comprehensive investigation of TiO2-Cu2O hetero-junction solar cells with different back contacts (Au, ITO, Cu or Ag). Combinatorial hetero-junction libraries consisting of a linear TiO2 thickness gradient produced by spray pyrolysis and a bell shaped Cu2O profile synthesized by pulsed laser deposition were chosen to investigate the impact of the two metal oxide layer thicknesses. The back contacts were deposited as round patches onto a grid of 13 × 13 points, 169 contacts for each contact material, forming a library containing 4 × 13 × 13 = 676 back contacts. Each back contact represented a solar cell with an individual TiO2 and Cu2O thickness. I-V measurements show that all four materials provide an ohmic contact and that the open circuit voltage of ∼300 mV is rather independent of both layer thicknesses and contact material. The size of the Cu2O crystals drastically decreases with distance from the center of deposition, which leads to a drastic increase of series resistance when the crystal size is <50 nm.

Effects of HCFC-123 exposure to maternal and infant rhesus monkeys on hepatic biochemistry, lactational parameters and postnatal growth.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

Effect of 4-vinylcyclohexene on micronucleus formation in the bone marrow of rats and mice.

This study was conducted to evaluate the potential of 4-vinylcyclohexene (VCH) to induce micronuclei in the bone marrow of mice and rats. Male and female Crl:CD BR (Sprague-Dawley) rats and B6C3F1/CrBR mice were exposed to VCH 6 hr/day for 2 days or for 13 weeks. In the 2-day study, mice were exposed by inhalation to 0, 250, 500, or 1000 ppm, and rats were exposed to 0, 500, 1000, or 2000 ppm. In the 13-week study, mice were exposed to 0, 50, 250, or 1000 ppm, and rats were exposed to 0, 250, 1000, or 1500 ppm. In each study, a separate group of mice was exposed to 1000 ppm 1,3-butadiene (BD) so that a comparison could be made between the two compounds. Likewise, cyclophosphamide was also included for rats as a positive control. Bone marrow was collected from VCH-exposed animals approximately 24 h and 48 h after the final exposure. There were no statistically significant increases in micronucleatedpolychromatic erythrocytes (MN-PCEs) among VCH-treated mice and rats at any dose level or sampling interval at either 2-days or 13-weeks. Also, no statistically significant differences in the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratios were observed in any of the VCH-treated mice and rats compared to air-exposed animals. As expected, both the butadiene-treated mice and the cyclophosphamide-treated rats showed significantly more MN-PCEs than the control animals.

Toxicity of tetrafluoroethylene and S-(1,1,2, 2-tetrafluoroethyl)-L-cysteine in rats and mice.

Groups of 25 female F344 rats and 25 female B6C3F1 mice were exposed to 0, 30, 300, 600, or 1200 ppm tetrafluoroethylene (TFE) by inhalation for up to 12 days. Another set of 25 female rats and 25 female mice of the same strains were given 0, 5, 20, or 50 mg/kg of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS) by oral gavage for 12 days. Both 12-day exposure regimens consisted of exposures for 5 consecutive days, a weekend with no exposures, and 4 consecutive daily exposures following the weekend. Five animals per group were sacrificed after the first exposure, the fifth exposure, and the ninth exposure for evaluation of cell proliferation in the liver and kidney. The remaining animals in each group (up to 10) were sacrificed after the ninth exposure (test day 12) for pathological evaluation of the liver, kidney, and spleen. Clinical pathology evaluations were performed on test day 11 or 12. Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice, but no histopathological changes in the liver. In the kidney, administration of TFE-CYS by gavage caused severe microscopic changes in rats, moderate-to-severe changes in mice, and no microscopic changes in the liver. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused increased liver weights and cell proliferation in the liver of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most pronounced after 5 days of exposure, and less evident or absent after 12 days of exposure. In the kidney, the cell proliferation and histopathologic response in rats was generally more pronounced than in mice. Kidney damage and cell proliferation were confined to the pars recta (P3) of the outer stripe of the outer medulla and medullary rays. Tubules in mice exposed to TFE and TFE-CYS had mostly regenerating cells by test day 12, while in rats the tubules still showed marked degeneration along with regeneration by the end of the study. The cortical labyrinth (P1 and P2 segments) was also affected at the 50 mg/kg dose of TFE-CYS in rats. Rats exposed to 50 mg/kg TFE-CYS had a mild anemia, and rats exposed to 1200 ppm TFE had slight, biologically inconsequential decreases in erythrocyte mass that may have been compound-related. In spite of the rather pronounced histopathologic changes in the kidneys of rats exposed to TFE-CYS, there was no clinical chemistry evidence for decreased kidney function. Increased levels of urinary fluoride were present in rats exposed to 300 ppm and greater of TFE, and in rats exposed to 20 and 50 mg/kg TFE-CYS. The spleen was not affected in this study. Overall, the results of this study suggest that effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-week exposure, as all effects that were seen with TFE were also seen with TFE-CYS.

1,1,1-Trifluoro-2,2-dichloroethane (HCFC-123) and 1,1,1-trifluoro-2-bromo-2-chloroethane (halothane) cause similar biochemical effects in rats exposed by inhalation for five days.

1,1,1-Trifluoro-2,2-dichloroethane (HCFC-123) and 1,1,1-trifluoro-2-bromo-2 chloroethane (halothane) are gases with anesthetic properties. HCFC-123 is used as a refrigerant, fire extinquishing agent, and solvent, while halothane is a clinical anesthetic. Much information is available on chronic toxicity of HCFC-123 in animals, while the information available for halothane is from short-term animal exposures or chronic, low level human exposures. Thus, there is little biochemical information available on similar endpoints for these two chemicals, which share common metabolites. In the present study, male rats were exposed to 5000 ppm HCFC-123, 5000 ppm halothane, or room air for 6 hr per day for 5 consecutive days. Rats exposed to both test compounds gained little or no weight during the study. Liver weights were slightly decreased in the rats exposed to HCFC-123 and halothane compared to controls. The serum triglycerides were decreased to approximately 20% of control level in rats exposed to both HCFC-123 and halothane, and serum cholesterol was decreased to less than 80% of control by both compounds. Both test compounds increased hepatic beta-oxidation by approximately 3-fold over control, and HCFC-123 caused a significant increase in hepatic cytochrome P450 content, while the increase in cytochrome P450 was not statistically significant in the halothane-treated rats. The results indicate that HCFC-123 and halothane share not only common metabolic pathways, but also several common biological effects, specifically those associated with peroxisome proliferation. These data indicate that human experience with halothane may be useful in the risk assessment of HCFC-123.

Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N-acetyl-L-cysteines.

The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/- 27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.

Subchronic nasal toxicity of hexamethylphosphoramide administered to rats orally for 90 days.

Rats were administered hexamethylphosphoramide (HMPA) at dosages of 10, 100, 300, and 1000 ppm in drinking water or at 15, 40, or 120 mg/kg/day by gavage for approximately 90 days. Another group of rats was implanted subcutaneously with HMPA-filled osmotic minipumps, designed to deliver a dosage of 40 mg/kg/day to prevent the possibility of direct contact of HMPA with the nasal epithelium. After 90 days at 10 ppm in the drinking water, some rats had tracheas lined with regenerated epithelium, but no HMPA-related lesions were present in any other organs and tissues. At 100 ppm, nasal lesions (epithelial denudation, regeneration, and squamous metaplasia) were mostly in the maxilloturbinates, tips of nasoturbinates, and the adjacent septum in the anterior nasal cavity (level I), but the lesions were confined to the ventral region of the mid-anterior nasal cavity (level II) and to recesses of the posterior nasal cavity (levels III and IV). At 300 ppm, nasal turbinates in level I were partially adhered to the nasal septum by fibrous tissue. In level II the lesions were mainly confined to the ventral medial meatus, but were scattered diffusely in levels III and IV. Denuded turbinates showed minimal bone proliferation. At 1000 ppm, the anterior nasal cavity was partially occluded by extensive adhesion of the turbinates to the nasal septum by granulation tissue and proliferating turbinate bone. The general architecture of the posterior nasal cavity was obliterated by the marked proliferation of turbinate bone and fibrous tissue in the interturbinate spaces. Tracheas showed regenerated epithelium and bronchi had focal epithelial denudation at 100, 300, and 1000 ppm. Foamy alveolar macrophages (histiocytosis) were increased in the lungs at 300 and 1000 ppm. Testicular atrophy occurred at 1000 ppm. No other tissues were affected by HMPA treatment. Nasal lesions in rats given HMPA by gavage were identical in nature to, but sometimes slightly more severe than, the lesions in rats given HMPA in the drinking water. Rats given 40 mg/kg/day HMPA via an osmotic minipump had slightly less severe nasal lesions than did the rats given the same dosage of HMPA by gavage. Testicular atrophy was present in the rats given 120 mg/kg/day by gavage. The results of this study show that, with the exception of bone proliferation, systemic delivery of HMPA or its metabolites to the nasal tissue following oral administration causes tissue damage similar to that caused by direct exposure of the nasal tissue via inhalation. Oral administration of HMPA is a less potent route for producing nasal lesions than is inhalation.

In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary.

4-Vinylcyclohexene (4-VCH), the dimer of 1,3-butadiene, is an ovarian toxicant in mice due to the formation of a diepoxide metabolite, but the tissue-specific site of formation of the metabolites is unknown. Microsomal preparations from liver, lung, and ovaries obtained from female Crl:CD BR rats and female B6C3F1 mice were tested for their ability to metabolize the following reactions: 4-VCH to 4-VCH-1,2-epoxide and 4-VCH-7,8-epoxide; 4-VCH-1,2-epoxide to 4-VCH diepoxide and 4-VCH-1,2-diol; 4-VCH-7,8-epoxide to 4-VCH diepoxide and 4-VCH-7,8-diol; and hydrolysis of 4-VCH diepoxide. Microsomes were incubated with the test chemical and the reaction products were analyzed by gas chromatography. Rat liver and lung microsomes and mouse liver and lung microsomes metabolized 4-VCH to 4-VCH-1,2-epoxide at detectable rates. Mouse liver had a Vmax for the reaction that was 56-fold higher than that for rat liver (11.1 and 0.20 nmol/min/mg protein, respectively). The Vmax for mouse lung was 2-fold higher than that for rat lung. 4-VCH-1,2-epoxide formation was not detected in ovarian microsomes from rats or mice. Metabolism of 4-VCH to 4-VCH-7,8-epoxide was detected in microsomes from rat liver and mouse liver and lung, at rates very low compared to those for metabolism to the 1,2-epoxide. Rat and mouse liver had very similar K(m) and Vmax values for metabolism of 4-VCH-1,2-epoxide to 4-VCH diepoxide. The Vmax for rat liver was 3.69 and for mouse liver was 5.35 nmol/min/mg protein. Rat and mouse ovaries did not have detectable capacity to metabolize 4-VCH-1,2-epoxide to the diepoxide. Rat and mouse liver and lung have very similar K(m) and Vmax values for metabolism of 4-VCH-7,8-epoxide to the diepoxide, while ovaries did not have detectable rates for this reaction. Hydrolysis of 4-VCH-1,2-epoxide to 4-VCH-1,2-diol was at similar rates in rat and mouse liver microsomes. Hydrolysis of 4-VCH-7,8-epoxide to 4-VCH-7,8-diol was detected only in rat liver microsomes. Hydrolysis of 4-VCH diepoxide was detected in rat and mouse liver and lung, and in rat ovary microsomes. The Vmax for rat liver was 9-fold greater than that for mouse liver (5.51 and 0.63 nmol/min/mg protein, respectively), and lung and ovary tissues were not as active as rat liver. The balance of activation versus detoxication reactions in rats and mice suggests that the mouse may be more susceptible to 4-VCH toxicity because of generation of high levels of epoxide metabolites. In general, the mouse is more efficient at metabolism of 4-VCH to epoxides than is the rat. In contrast, the rat may be more efficient at hydrolysis of epoxides. Thus, the rat would tend to produce a lower concentration of epoxide metabolites than the mouse, at equal doses of 4-VCH.

Pharmacokinetics and metabolism of vinyl fluoride in vivo and in vitro.

Vinyl fluoride (VF) is an inhalation carcinogen at concentrations of 25 ppm or greater in rats and mice. The main neoplastic lesion induced in rodents was hepatic hemangiosarcomas, and mice were more sensitive than rats. In a first set of experiments, groups of three rats or five mice were exposed to VF in a closed-chamber gas uptake system at starting concentrations ranging from 50 to 250 ppm. Chamber concentrations of VF were measured every 10-12 min by gas chromatography. Partition coefficients were determined by the vial equilibration technique and used as parameters for a physiologically based pharmacokinetic (PBPK) model. Mice showed a higher whole-body metabolic capacity compared to rats (Vmax = 0.3 vs 0.1 mg/hr-kg). Both species had an estimated Km of < or = 0.02 mg/liter. The specificity for the oxidation of VF in vivo was determined by selective inhibition or induction of CYP 2E1. Inhibition with 4-methylpyrazole completely impaired VF uptake in rats and mice, whereas induction with ethanol (rats only) increased the metabolic capacity by two- to threefold. The pharmacokinetics of VF were also investigated in vitro. Microsomes from rat and mouse liver were incubated in a sealed vial with VF and an NADPH-regenerating system. Headspace concentrations (10-300 ppm) were monitored over time by gas chromatography. Consistent with the in vivo data, VF was metabolized faster by mouse microsomes than by rat microsomes (Vmax = 3.5 and 1.1 nmol/hr-mg protein, respectively). The rates of metabolism by human liver microsomes were generally in the same range as those found with rat liver microsomes (Vmax = 0.5-1.3 nmol/hr-mg protein), but one sample was similar to mice (Vmax = 3.3 nmol/ hr-mg protein). Metabolic rates in human microsomes were found to correlate with the amount of CYP 2E1 as determined by Western blotting and by chlorzoxazone 6-hydroxylation. It is concluded that the greater metabolic capacity of mice for VF both in vivo and in vitro may contribute to their greater susceptibility to tumor formation. CYP 2E1 is clearly the main isozyme involved in the oxidation of VF in all species tested. VF pharmacokinetics and metabolism in humans may depend upon the interindividual variability in the expression level of CYP 2E1. The excellent correspondence between in vivo and in vitro kinetics in rodents improves. substantially the degree of confidence for human in vivo predictions from in vitro data.

Fluoroacetate-mediated toxicity of fluorinated ethanes.

A series of 1-(di)halo-2-fluoroethanes reported in the literature to be nontoxic or of low toxicity were found to be highly toxic by the inhalation route. Experiments were performed that showed the compounds, 1,2-difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane to be highly toxic to rats upon inhalation for 4 hr. All four compounds had 4-hr approximate lethal concentrations of < or = 100 ppm in rats. In contrast, 1,1-difluoroethane (commonly referred to as HFC-152a) has very low acute toxicity with a 4-hr LC50 of > 400,000 ppm in rats. Rats exposed to the selected toxic fluoroethanes showed clinical signs of fluoroacetate toxicity (lethargy, hunched posture, convulsions). 1,2-Difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane were shown to increase concentrations of citrate in serum and heart tissue, a hallmark of fluoroacetate intoxication. 19F NMR analysis confirmed that fluoroacetate was present in the urine of rats exposed to each toxic compound. Fluorocitrate, a condensation product of fluoroacetate and oxaloacetate, was identified in the kidney of rats exposed to 1,2-difluoroethane. There was a concentration-related elevation of serum and heart citrate in rats exposed to 0-1000 ppm 1,2-fluoroethane. Serum citrate was increased up to 5-fold and heart citrate was increased up to 11-fold over control citrate levels. Metabolism of 1,2-difluoroethane by cytochrome P450 (most likely CYP2E1) is suspected because pretreatment of rats or mice with SKF-525F, disulfiram, or dimethyl sulfoxide prevented or delayed the toxicity observed in rats not pretreated. Experimental evidence indicates that the metabolism of the toxic fluoroethanes is initiated at the carbon-hydrogen bond, with metabolism to fluoroacetate via an aldehyde or an acyl fluoride. The results of these studies show that 1-(di)halo-2-fluoroethanes are highly toxic to rats and should be considered a hazard to humans unless demonstrated otherwise.

Finding the global minimum: a fuzzy end elimination implementation.

The 'fuzzy end elimination theorem' (FEE) is a mathematically proven theorem that identifies rotameric states in proteins which are incompatible with the global minimum energy conformation. While implementing the FEE we noticed two different aspects that directly affected the final results at convergence. First, the identification of a single dead-ending rotameric state can trigger a 'domino effect' that initiates the identification of additional rotameric states which become dead-ending. A recursive check for dead-ending rotameric states is therefore necessary every time a dead-ending rotameric state is identified. It is shown that, if the recursive check is omitted, it is possible to miss the identification of some dead-ending rotameric states causing a premature termination of the elimination process. Second, we examined the effects of removing dead-ending rotameric states from further considerations at different moments of time. Two different methods of rotameric state removal were examined for an order dependence. In one case, each rotamer found to be incompatible with the global minimum energy conformation was removed immediately following its identification. In the other, dead-ending rotamers were marked for deletion but retained during the search, so that they influenced the evaluation of other rotameric states. When the search was completed, all marked rotamers were removed simultaneously. In addition, to expand further the usefulness of the FEE, a novel method is presented that allows for further reduction in the remaining set of conformations at the FEE convergence. In this method, called a tree-based search, each dead-ending pair of rotamers which does not lead to the direct removal of either rotameric state is used to reduce significantly the number of remaining conformations. In the future this method can also be expanded to triplet and quadruplet sets of rotameric states. We tested our implementation of the FEE by exhaustively searching ten protein segments and found that the FEE identified the global minimum every time. For each segment, the global minimum was exhaustively searched in two different environments: (i) the segments were extracted from the protein and exhaustively searched in the absence of the surrounding residues; (ii) the segments were exhaustively searched in the presence of the remaining residues fixed at crystal structure conformations. We also evaluated the performance of the method for accurately predicting side chain conformations. We examined the influence of factors such as type and accuracy of backbone template used, and the restrictions imposed by the choice of potential function, parameterization and rotamer database. Conclusions are drawn on these results and future prospects are given.

A strategy for characterization of polyethylene glycol-derivatized proteins. A mass spectrometric analysis of the attachment sites in polyethylene glycol-derivatized superoxide dismutase.

Base treatment of polyethylene glycol-derivatized superoxide dismutase in which the polyethylene glycol is linked to the protein via a succinyl bridge, removes the polyethylene glycol leaving a succinyl marker. Exhaustive succinylation with d4-succinic anhydride completes the derivatization in order to minimize fractionation in proteolysis, chromatography, and desorption in the mass spectrometer. Production of peptides from the derivatized protein for high resolution and high-resolution tandem MS allows identification of the site that had been derivatized by polyethylene glycol and the determination of the amount of polyethylene glycol originally at each site. The mass spectrometric strategy outlined herein can be applied to other proteins derivatized for therapeutic administration.

Multipole correction of atomic monopole models of molecular charge distribution. I. Peptides.

The defects in atomic monopole models of molecular charge distribution have been analyzed for several model-blocked peptides and compared with accurate quantum chemical values. The results indicate that the angular characteristics of the molecular electrostatic potential around functional groups capable of forming hydrogen bonds can be considerably distorted within various models relying upon isotropic atomic charges only. It is shown that these defects can be corrected by augmenting the atomic point charge models by cumulative atomic multipole moments (CAMMs). Alternatively, sets of off-center atomic point charges could be automatically derived from respective multipoles, providing approximately equivalent corrections. For the first time, correlated atomic multipoles have been calculated for N-acetyl, N'-methylamide-blocked derivatives of glycine, alanine, cysteine, threonine, leucine, lysine, and serine using the MP2 method. The role of the correlation effects in the peptide molecular charge distribution are discussed.

Histochemical localization of formaldehyde dehydrogenase in the rat.

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowman's and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.

Effect of sulfite on the covalent reaction of benzo[a]pyrene metabolites with DNA.

Sulfur dioxide (SO2) potentiates the carcinogenicity of polycyclic aromatic hydrocarbons. To investigate the mechanism of SO2 cocarcinogenesis, the effect of sulfite, the hydrated form of SO2, on the covalent reaction of benzo[a]pyrene (BaP) metabolites with DNA in vitro was measured. [14C]BaP was incubated with rat lung or liver post-mitochondrial supernatant (S9), an NADPH generating system, calf thymus DNA and sodium sulfite (0-20 mM). In the presence of lung S9, covalent reaction increased linearly from 0.66 to 1.20 pmol BaP metabolites per mg DNA with increasing sulfite concentrations. Addition of sulfite to rat liver S9 also increased BaP-DNA adduct formation with BaP-DNA adducts increasing from 80 to 120 pmol per mg DNA. Sulfite altered the amount and pattern of BaP metabolites formed by either lung or liver enzyme preparations. BaP was metabolized more extensively and the amount of water soluble BaP metabolites formed increased significantly with sulfite present. With lung S9, the amount of BaP-tetrols, diols, and phenols increased slightly. With liver S9, diol and phenol formation was significantly lower while tetrol formation was unchanged. Incubation of rat lung S9 with sulfite resulted in formation of glutathione S-sulfonate (GSSO3H), a known inhibitor of glutathione S-transferases mediating the conjugation of glutathione (GSH) and BaP epoxides. Our results suggest that sulfite may, by altering the overall metabolic activation and detoxication of BaP, or by reacting directly with DNA, subsequently affect the covalent reaction of BaP metabolites with DNA. These are offered as possible mechanisms to explain the cocarcinogenic effect of SO2.

Effects of sulfite on glutathione S-sulfonate and the glutathione status of lung cells.

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.

Mechanistic studies on chloral toxicity: relationship to trichloroethylene carcinogenesis.

Chloral (trichloroacetaldehyde), the major metabolite of trichloroethylene (TCE), was investigated for its potential to form DNA-protein cross-links (DPX), a lesion produced by other aldehydes. Chloral did not form DPX in rat liver nuclei at concentrations up to 250 mM for 30 min at 37 degrees C, while chloroacetaldehyde (47 mM) and acetaldehyde (200 mM) did form cross-links. Experiments with the aldehyde-trapping reagents thiosemicarbazide and semicarbazide showed that chloral did not react, in contrast with aldehydes that form DPX. This indicates a very strong hydration of chloral. Mice given 800 mg/kg [14C]chloral after pretreatment with 1500 mg/kg TCE for 10 days had no detectable covalent binding of 14C to DNA in the liver. These results do not support a genotoxic theory of carcinogenesis for TCE mediated through chloral.

Covalent reactions in the toxicity of SO2 and sulfite.

Toxic effects of SO2 and sulfite such as bronchitis and bronchoconstriction have been well documented. SO2 has also been suggested to potentiate carcinogenic effects of PAH. However, the molecular basis of these toxic effects is unclear. We have examined the covalent reaction of SO2 and sulfite with cellular proteinacious and nonproteinaceous sulfhydryl compounds using rat liver, and lung and human lung derived A549 cells. Reactions of sulfite and protein in rat and human lung cells reveals at least three proteins with sulfite-reactive disulfide bonds. Besides fibronectin and serum albumin, which had been reported to contain sulfonated products following exposure to sulfite, we have found one other protein with sulfite-binding capabilities. Since the integrity of disulfide bonds is crucial to the tertiary structure and thus protein function, the disruption of protein structure by sulfitolysis may result in altered cellular activities leading to biochemical lesions. Using carefully controlled conditions, reproducible GSH contents can be found in cultured cells and used as an experimental basis for studying alterations in the GSH and GSSG content of cells. Sulfitolysis of GSSG results in the formation of GSSO3H in A549 cells, and possibly in the lung. GSSO3H can be reduced enzymatically by GSSG reductase. However, the Km of GSSO3H is high compared to that of GSSG, suggesting the existence of a transient concentration of GSSO3H once it is formed. Cysteine S-sulfonate is, however, not reduced by cytosolic extracts in the presence of NADPH and would have to be eliminated from the cell by other means. GSSO3H is a strong competitive inhibitor of GST in rat liver and lung and A549 cells, using 1-chloro-2,4-dinitrobenzene as a substrate. It also inhibits the formation of GSH conjugates of BP 4,5-oxide, anti and syn BPDE, but to a lesser extent. These results suggest that SO2 may affect the detoxification of xenobiotic compounds by inhibiting, via formation of GSSO3H, the enzymatic conjugation of GSH and reactive electrophiles. Since GSH conjugation represents the major pathway of elimination of BP epoxides in the lung, our results offer a possible explanation for the cocarcinogenicity of SO2 with PAHs. These data suggest that the sulfitolysis reaction of sulfite is the common reaction mechanism mediating the underlying biochemical reactions leading to both the toxic and cocarcinogenic properties of SO2. Quantitation of sulfitolysis products and their interaction with cellular processes should provide a coherent scheme relating SO2 and sulfite toxicity among animal species and humans.

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography.

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.