Transforming an EPA QA/R-2 quality management plan into an ISO 9002 quality management system.

The Environmental Protection Agency's (EPA) Office of Emergency and Remedial Response (OERR) requires environmental data of known quality to support Superfund hazardous waste site projects. The Quality Assurance Technical Support (QATS) Program is operated by Shaw Environmental and Infrastructure, Inc. to provide EPA's Analytical Operations Center (AOC) with performance evaluation samples, reference materials, on-site laboratory auditing capabilities, data audits (including electronic media data audits), methods development, and other support services. The new QATS contract awarded in November 2000 required that the QATS Program become ISO 9000 certified. In a first for an EPA contractor, the QATS staff and management successfully transformed EPA's QA/R-2 type Quality Management Plan into a Quality Management System (QMS) that complies with the requirements of the internationally recognized ISO 9002 standard and achieved certification in the United States, Canada, and throughout Europe. The presentation describes how quality system elements of ISO 9002 were implemented on an already existing quality system. The psychological and organizational challenges of the culture change in QATS' day-to-day operations will be discussed for the benefit of other ISO 9000 aspirants.

Studies on the mechanism of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)-induced hepatotoxicity. III. Ultrastructural characterization of bile duct injury.

The antineoplastic nitrosourea CCNU is a known hepatotoxin which has been shown to cause hyperbilirubinemia and reduction in bile flow. We studied morphological alterations in the common bile duct and interlobular bile ducts at 6, 12, and 24 h in male rats given a single oral dose (50 mg/kg) of CCNU. The portal vein was perfused with 1.0% glutaraldehyde fixative. Portal areas and the common bile duct were selectively dissected and processed using standard methods for light and transmission electron microscopy. The epithelial cells of larger common bile duct and interlobular bile ducts showed increased rough endoplasmic reticulum, markedly increased free ribosomes, and mitochondrial degeneration at 6 and 12 h after CCNU. There was also bile imbibition and loss of microvilli, which increased in severity at 12 and 24 h. The interstitium showed infiltration by acute inflammatory cells and dilated capillaries at 6 h. By 24 h, degeneration of epithelial cells was extensive; cells became necrotic and sloughed into the duct lumen. The smaller bile ductules showed no significant degenerative changes; adjacent hepatocytes were unremarkable. Early CCNU injury appears localized in the large bile ducts and reflects inflammatory edema, bile stasis, and degeneration of epithelial cells. Our studies suggest that this ductal injury may reflect metabolism of CCNU to reactive species within the bile ducts.

Early effects of 1,1-dichloroethylene on canalicular and plasma membranes: ultrastructure and stereology.

The pathogenesis of 1,1-dichloroethylene (1,1-DCE)-induced hepatotoxicity was investigated in fasted male rats by identifying the earliest morphological alterations in organelles. In situ perfusion-fixed liver tissue was examined by light and electron microscopy at 1, 2, or 3 hr after oral administration of 25, 50, and 100 mg 1,1-DCE/kg in mineral oil. The earliest morphological alterations, which occurred within 1 to 2 hr after 1,1-DCE administration, were dilation of bile canaliculi with an increase in the number of microvilli or membrane fragments in canaliculi and the formation of canalicular diverticuli in centrolobular hepatocytes. Subsequently, microvilli on sinusoidal surfaces were disrupted or lost. Membrane whorls were frequently found in bile canaliculi, the space of Disse, and between the lateral membranes of hepatocytes at early times. As injury progressed, centrolobular hepatocytes retracted from endothelial cells and sinusoidal plasma membranes invaginated to form cytoplasmic vacuoles. Stereological analysis of centrolobular hepatocytes at the 25 mg/kg dose showed a significant increase in canalicular volume density by 3 hr and no detectable alteration in mitochondrial volume density. These results indicate that changes in canalicular shape and microvilli configuration are the earliest morphological alterations following 1-DCE ingestion.

Lung injury and repair: DNA synthesis following 1,1-dichloroethylene.

Injury and cellular proliferation in the lung were examined following administration of 1,1-dichloroethylene (1,1-DCE) or vinylidene chloride. C57BL/6 male mice were treated orally with 200 mg/kg of 1,1-DCE prior to a single pulse of tritiated thymidine [( 3H]TdR). Necrosis and exfoliation of Clara cells of bronchiolar epithelium were evident by 1 day after chemical administration, and increased in severity by 2 days. A regenerative response was observed at 3 days after 1,1-DCE administration, and by 7 days the epithelium was substantially restored. At 30 days after 1,1-DCE, re-epithelization was achieved and areas devoid of epithelium were not observed. Changes in cellular proliferation were calculated from measurements of [3H]TdR incorporation into total pulmonary DNA. Activity of [3H]TdR was significantly inhibited at 1 day after chemical administration and thereafter increased: a peak of synthesis occurred between 3 and 5 days. At 7 days after 1,1-DCE administration, incorporation of [3H]TdR decreased to levels that were not significantly different from those of control animals. Autoradiographic examination of 0.5 micron thick plastic-embedded lung sections showed that [3H]TdR was incorporated into the DNA of bronchiolar epithelial cells, macrophages, interstitial, endothelial and Type II alveolar cells. However, the majority of the label was taken up by the nonciliated bronchiolar epithelial cells. The increased [3H]TdR incorporation into whole lung correlated with repopulation of bronchioles which was observed following injury. The results demonstrated that 1,1-DCE-induced damage to Clara cells of the bronchiolar epithelium was severe and rapid; re-epithelization was achieved in a relatively short time whereas differentiation was a prolonged process.

Adsorption and reversed-phase high-performance liquid chromatography of p-nitrobenzyl esters of monohydroxy fatty acids.

To enhance the UV detectability of hydroxy fatty acids, p-nitrobenzyl (PNB) esters of twenty-two different monohydroxy fatty acids of various chain-lengths (C16-C22) and differing positional isomers were formed using O-(p-nitrobenzyl)-N,N-(diisopropyl)-isourea (PNBDI) as alkylating agent. Reversed-phase and adsorption high-performance liquid chromatography (HPLC) of the twenty-two monohydroxy fatty acid PNB esters were studied. The PNB group did not dominate the chromatographic properties of monohydroxy fatty acids and it did not interfere with the HPLC separation of positional isomers. PNBDI was, however, found to be less than ideal for formation of PNB derivatives of monohydroxy fatty acids because UV absorbing contaminants of PNBDI interfered with the HPLC analyses.

Cholestasis and increased biliary excretion of inulin in rats given 1,1-dichloroethylene.

Bile flow and biliary excretion of the inert solute [3H]inulin were monitored in unanesthetized, freely moving male rats for 4 h after oral administration of 1,1-dichloroethylene (1,1-DCE) at a dose of 200 mg/kg. Comparisons were made between 4 groups: fed-controls, fed-1,1-DCE treated, fasted-controls, and fasted-1,1-DCE treated. Biliary inulin excretion was assessed at 30-min intervals as total excretion and as bile/plasma ratio. 1,1-DCE treatment was consistently associated with at least a 2-fold increase in both parameters of inulin excretion within 2 h after toxin administration. In contrast, 1,1-DCE treatment was not associated with changes in plasma inulin values at any time or in liver/plasma inulin ratios at 4 h. Bile flow decreased in all groups: gradually by 30% in the fed and fasted controls, by 40% in the fed-1,1-DCE treated group, and markedly by 65% in the fasted-1,1-DCE treated group. Liver damage at 4 h as reflected by elevated plasma activities of liver-derived enzymes was found only in fasted-1,1-DCE treated rats. Thus the cholestatic effect of 1,1-DCE appears related to the development of liver damage whereas other aspects of the hepatic response to 1,1-DCE may enhanced biliary excretion of inulin.

Rapid, substrate-specific, and dose-dependent deactivation of liver cytosolic glutathione S-transferases in vivo by 1,1-dichloroethylene.

Administration of 200 mg 1,1-dichloroethylene (1,1-DCE)/kg to fasted male rats rapidly decreased liver cytosolic glutathione (GSH) S-transferase activities by half within 1 hr. This early decrease was not associated with increased serum activities of this soluble enzyme and is considered due to enzyme deactivation. The early decrease in enzyme activities was concomitant with a three-fourths depletion of cytosolic GSH and preceded changes in cytochrome P-450 and the onset of liver cytotoxicity, both of which occurred abruptly between 2 and 3 hr. Substantial changes in GSH S-transferase activities at 4 hr were produced only by severely hepatotoxic doses of 1,1-DCE. The early decrease in hepatic GSH S-transferase activities was selective for substrates dichloronitrobenzene, chlorodinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)-propane with apparent sparing of activity towards ethacrynic acid. The rapid, selective and dose-dependent deactivation of the hepatic GSH S-transferases could be relevant to the catastrophic hepatotoxicity of 1,1-DCE.

Metabolism of [14C]carbon tetrachloride to exhaled, excreted and bound metabolites. Dose-response, time-course and pharmacokinetics.

Fasted male rats were given six doses of 14CCl4 ranging from non-hepatotoxic (0.1 mmole/kg) to severely hepatotoxic (26 mmoles/kg). Time-course and pharmacokinetics of CCl4, 14CO2 and CHCl3 elimination by exhalation were monitored by measuring amounts recovered in breath during discrete 15-min intervals for 8-12 hr. Amounts of 14C-labeled metabolite recovered bound to liver macromolecules at 24 hr and excreted in urine or feces for 24 hr were also determined. Comparison pharmacokinetic studies were done with 14CHCl3 and Na(2)14CO3. After all doses of 14CCl4, the major metabolite was CO2, twenty to thirty times less metabolite was recovered bound to liver macromolecules, and intermediate amounts of metabolite were excreted in urine and feces. CHCl3 was the least abundant metabolite at low CCl4 doses, but the second most abundant at high doses. Stronger associations were found between the magnitude of liver injury at 24 hr (quantitated as serum glutamate-pyruvate transaminase activity) and the extent or rate of CCl4 metabolism by pathways leading to CO2 and CHCl3 than by pathways leading to 14C-metabolites bound in liver or excreted in urine. Time-course and pharmacokinetic data indicated that a major pathway of CCl4 metabolism leading to CO2 became impaired within 2 hr after administration of hepatotoxic doses of CCl4.

1,1-Dichloroethylene: an apoptotic hepatotoxin?

Within 2 hr after 1,1-dichloroethylene administration, the following phenomena occur in livers of fasted rats: dilation and disruption of bile canaliculi, plasma membrane invagination and loss of microvilli, cytoplasmic vacuolation, and loss of density in mitochondrial matrices. Early, selective loss of enzyme activities was localized by histochemical staining to bile canalicular, and inner and outer mitochondrial membranes. Biliary permeability to inulin increased, a change suggestive of the breakdown of junctions between hepatocytes. Endoplasmic reticulum and lysosomes appeared spared. In addition, scattered, individual hepatocytes exhibited changes characteristic of apoptosis by 2 hr: chromatin aggregation and margination, nucleolar coarse granulation and enlargement, rounded blebs and proturberances on cell surfaces, and the separation of these cells from surrounding parenchyma. In contrast, evidence of plasma membrane leakiness to K+, Ca2+ and soluble cytoplasmic enzymes was not detected until after 2 hr. Based on these observations, we propose that 1,1-dichloroethylene may initiate apoptosis-like cell degradation in selected parenchymal cells prior to or coincident with centrolobular necrosis.

A single step method for the separation of rat liver cytosolic glutathione S-transferase isozymes.

A simple, single step chromatographic method was developed to separate the liver cytosolic glutathione S-transferase (GSH-S-t) isozymes from each other and from the bulk of the cytosolic protein. Five peaks of GSH-S-t activity, tested with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, were eluted. By comparison of the activities with CDNB and the other substrate 3,4-dichloronitrobenzene (DCNB) the five peaks could be identified as GSH-S-t isozymes C, B, A, and AA, being GSH-S-t isozyme C eluted in two different peaks. The method was used to detect a decrease of specific GSH-S-t isozymes in the cytosol of rats intoxicated with carbon tetrachloride, as compared with control rats.

Free radicals and disease in man.

Free radicals and related activated electronic species are produced in biological systems in antimicrobial defense, through the action of the mixed function monooxygenases, by various oxidative enzymes such as xanthine oxidase, and by autooxidations mediated by such agents as heavy metals or quinones. While the evidence is circumstantial, excessive unconfined or inappropriate production of radical species in inflammation, the metabolism of exogenous chemicals, or through autooxidation probably plays a significant role in human disease.

Relationships between the pharmacokinetics of carbon tetrachloride conversion to carbon dioxide and chloroform and liver injury.

Rate and extent of CCl4 metabolism by pathways leading to CO2 and CHCl3 were evaluated by measuring the amounts of these metabolites exhaled during discrete intervals following six different doses of CCl4. Pulmonary pharmacokinetics of 14CO2 and CHCl3 exhalation after CCl4 administration were compared with those after Na214CO3 and 14CHCl3 administration. Exhalation of 14CO2 metabolite declined more rapidly than expected after hepatotoxic doses of CCl4. This decline could be due to injury associated changes in the metabolism of CCl4.

Lactate dehydrogenase activity in mouse lung following 1,1-dichloroethylene: index of airway injury.

Lactate dehydrogenase (LD) activity and its isozyme profile in mouse lung homogenate was affected by oral administration of 1,1-dichloroethylene (1,1-DCE). Following 100 mg 1,1-DCE/kg, LD-3 increased significantly. After 200 mg 1,1-DCE/kg, LD-5 increased whereas LD-1 and LD-2 decreased, with a resultant higher M:H ratio than controls. In contrast, elevated LD activity in serum following 1,1-DCE was predominantly associated with striking increases in total activity and changes in isozyme patterns resulting in a decrease in the M:H ratio. LD activity in liver and erythrocytes were unaffected by 1,1,-DCE administration. Although total activity in kidney was decreased, no changes were detected in the isozyme profile. Pulmonary damage induced by 1,1-DCE was reflected in significant increases in total activity and all isozymes in bronchopulmonary lavage fluids. Thus, detection of lung-derived LD activity in lung lavage fluids can be a useful index of pulmonary airway injury.

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4.

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.

Structure-activity relations between alkyl nucleophilic chemicals causing duodenal ulcer and adrenocortical necrosis.

Structure-activity relationships were qualitatively and quantitatively examined for 56 chemicals (e.g., derivatives of propionitrile, acrylonitrile and cysteamine) which caused duodenal ulcer and/or adrenocortical necrosis in rats. For the first time the duodenal ulcerogenic property of numerous chemicals has been studied in a rational and predictive manner. Ulcerogenic activity was most intense in the carbonitriles attached to two or three carbon backbones and diminished by shortening, lengthening, branching, unsaturating, halogenating or hydroxylating the carbon chains. Different modes of action are implied. Adrenocorticolytic potency was associated with unsaturation of the carbon chain and substitution of the nitrile by thiol or amine radicals. An action of these chemicals on the central nervous system has been suggested.

Isopropanol enhancement of carbon tetrachloride metabolism in vivo.

We examined the effects of isopropanol (ISOP) pretreatment on the metabolism of 14CCl4 to 14CO2 and CHCl3 exhaled in the breath, to 14C metabolite excreted in 24 hr urine and feces from 0 to 24 hr, and to 14C metabolite bound to liver at 24 hr. Fasted male rats were given 0.1 or 2.0 mmoles 14CCl4/kg. ISOP pretreatment, which markedly enhanced the hepatotoxicity of CCl4, selectively enhanced the rate and total extent of 14CO2 and CHCl3 metabolite exhalation. The pathways of CCl4 metabolism leading to CO2 and CHCl3 metabolite formation may be more relevant to the hepatotoxicity of CCl4 than the pathways leading to urinary, fecal or covalently bound metabolites.