Approaches to characterizing human health risks of exposure to fibers.

Naturally occurring and man-made (synthetic) fibers of respirable sizes are substances that have been identified by the U.S. Environmental Protection Agency (U.S. EPA) as priority substances for risk reduction and pollution prevention under the Toxic Substances Control Act (TSCA). The health concern for respirable fibers is based on the link of occupational asbestos exposure and environmental erionite fiber exposure to the development of chronic respiratory diseases, including interstitial lung fibrosis, lung cancer, and mesothelioma in humans. There is also considerable laboratory evidence indicating that a variety of fibers of varying physical and chemical characteristics can elicit fibrogenic and carcinogenic effects in animals under certain exposure conditions. This paper discusses key scientific issues and major default assumptions and uncertainties pertaining to the risk assessment of inhaled fibers. This is followed by a description of the types of assessment performed by the U.S. EPA to support risk management actions of new fibers and existing fibers under TSCA. The scope and depth of these risk assessments, however, vary greatly depending on whether the substance under review is an existing or a new fiber, the purpose of the assessment, the availability of data, time, and resources, and the intended nature of regulatory action. In general, these risk assessments are of considerable uncertainty because health hazard and human exposure information is often incomplete for most fibers. Furthermore, how fibers cause diseases and what specific determinants are critical to fiber-induced toxicity and carcinogenicity are still not completely understood. Further research to improve our knowledge base in fiber toxicology and additional toxicity and exposure data gathering are needed to more accurately characterize the health risks of inhaled fibers.

Evaluation of reduced protocols for carcinogenicity testing of chemicals: report of a joint EPA/NIEHS workshop.

The current U.S. Environmental Protection Agency (EPA) and other national/international guidelines specify the use of two species and two sexes rodents (usually the rat and the mouse) for carcinogenicity testing of chemicals. In view of the enormous number of chemicals to be tested, the high cost of testing, and the large number of animals used in the present protocol, many academic, industrial, and government authorities are examining the possibility of using a reduced protocol (less than two species and two sexes of rodents) for carcinogenicity testing of chemicals. The use of a reduced protocol offers many advantages as well as some disadvantages. To pursue further the potential implications and impacts of using a reduced protocol for carcinogenicity testing on the processes of hazard identification and risk assessment, a workshop entitled "Evaluation of Reduced Protocols for Carcinogenicity Testing of Chemicals" was held at the Embassy Suites Hotel in Alexandria, Virginia on September 22 and 23, 1992. It was cosponsored by EPA's Office of Prevention, Pesticides and Toxic Substances (OPPTS) and the National Toxicology Program of the National Institutes of Environmental Health Sciences (NTP/NIEHS) and attended by more than 60 participants from government, industry, academia, and the general public. The Expert Consensus Panel and most of the participants supported the use of reduced protocols in carcinogenicity testing. However, it was recognized that reduced protocols may not be appropriate for the testing of all chemicals and that additional analyses/data may be needed for selection of the most appropriate reduced protocol for certain chemicals/chemical classes.

Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz[a]anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects.

The leukemogens 7,12-dimethylbenz[a]anthracene (DMBA) and 7,8,12-trimethylbenz[a]anthracene (TMBA) bind covalently in vivo to DNA of Long-Evans rats in the hematopoietic organs, spleen and bone marrow, and in the liver, a non-target organ. Both TMBA and DMBA depleted bone marrow cells and both agents bound persistently to the DNA of bone marrow and of liver, and less to that of spleen. The three main DMBA:deoxyribonucleoside adducts in spleen, bone marrow and liver were the same as those found previously in the liver (Dipple et al. (1983) Cancer Res., 43, 4132). There were no organ-specific or age-dependent differences in the relative amounts of adducts formed. There appears to be no direct correlation between the susceptibility of an organ to carcinogenesis and the nature and relative amount of the specific adducts formed, at least for the three organs studied here.

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo.

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.

Enhanced nitrosourea cytotoxicity in cell culture by sodium butyrate.

In HeLa S3 cells, sodium butyrate was found to potentiate the cytotoxicity of chloroethylnitrosoureas and alkylating agents in vitro. Using a soft-agar colony-forming assay, 2.5 and 5.0 mM sodium butyrate pretreatment for 22 h increased the cell killing efficacy of both methyl- and chloroethylnitrosoureas by between 30 and 70%. The potentiation of cytotoxicity of bifunctional nitrogen mustards by butyrate was less than that of nitrosoureas, with a 15-30% increased cell kill at 5 mM butyrate. Sodium butyrate per se reduced plating efficiency and caused growth delay if residual levels (calculated at 100 microM for starting concentrations of 5 mM) were not removed by washing prior to plating.

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene.

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) have previously been shown to induce dose-dependent DNA strand breaks in primary hepatocytes from mice and rats. In an attempt to determine the relationship between the extent of DNA strand breaks and the formation of specific DNA-carcinogen bound adducts in murine liver, the capability of N-OH-AAF and N-OAc-AAF to induce both DNA single strand breaks and adduct formation in in vivo and in primary hepatocytes was measured. N-OH-AAF induced a low level of DNA damage in F344 rats (10 mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment. The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene (Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene (Gua-C8-AF) 34% versus 67% and 3-(guanin-N2-yl)-2-acetylaminofluorene (Gua-N2-AAF) 11% versus 10%, respectively, for rat and mouse liver. An additional unknown adduct (12%) was detected in mouse liver. Dose dependent DNA binding and formation of individual DNA adducts were observed in rat and mouse primary hepatocytes following 1 h exposure to [ring-3H]-N-OH-AAF (0.1-20 microM) and [ring-3H]-N-OAc-AAF (5-20 microM). The patterns of DNA adducts in mouse and rat primary hepatocytes exposed to N-OH-AAF and N-OAc-AF were similar to those obtained in liver following in vivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon (10(-4) M) completely inhibited DNA damage induced by N-OH-AAF in mouse and partially in rat hepatocytes while DNA damage caused by N-OAc-AAF was only partially inhibited by paraoxon (10(-4) M) in both species. Parallel experiments showed that paraoxon, at low concentration (10(-6) M), did not alter either the level of DNA binding or the pattern of adduct formation in rat hepatocytes treated with N-OH-AAF (20 microM). However, at 10(-4) M paraoxon partially blocked DNA binding (60%) and the formation of Gua-C8-AAF (95%) and Gua-N2-AAF (80%) while Gua-C8-AF was increased two-fold. In mouse hepatocytes paraoxon pretreatment (10(-4) M) inhibited the formation of Gua-C8-AF by 70% following exposure to N-OH-AAF (20 microM). Gua-C8-AAF and Gua-N2-AAF were also inhibited but only at 10(-4) M paraoxon.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions of phenobarbital with propranolol in the dog. 2. Bioavailability, metabolism and pharmacokinetics.

This article describes the effects of chronically administered phenobarbital (180 mg/day p.o.) in dogs on the bioavailability of single 40-mg oral doses of propranolol and on the pharmacokinetics and the patterns of propranolol metabolism in urine after both oral (40 mg) and i.v. (6 mg) doses. Phenobarbital decreased the bioavailability of propranolol from 7.7 to 3.5%. By using a gas chromatography/mass spectrometry technique, the quantitative metabolic pattern of i.v. propranolol was unaltered by phenobarbital, but increases in hydroxylation and induction of new metabolites were observed for oral propranolol after phenobarbital treatment. Chronic phenobarbital treatment led to significant decreases in the half-lives of propranolol after both i.v. and oral doses. Little change was observed in the hepatic blood flow, systemic clearance or intrinsic oral clearance. These data demonstrate the completely nonrestrictive elimination of propranolol. With phenobarbital, both the extent of plasma protein binding and the distribution into erythrocytes change significantly, yet the overall clearance remains unaltered. The half-lives fell in responses to the binding-induced reduction in the apparent volume of distribution.

Effects of dl-propranolol on lidocaine disposition in the perfused rat liver.

The effects of flow rate and dl-propranolol pretreatment on the hepatic clearance of lidocaine were studied in the perfused rat liver. Livers were perfused in situ with Krebs' bicarbonate buffer (pH 7.4) containing 35 muM of nonlabeled lidocaine and trace amounts of 14C-labeled lidocaine for up to 60 min at 14 or 7 ml/min. Lidocaine was rapidly eliminated from the perfusate at 14 ml/min but was at slower rate at 7 ml/min. Pretreatment of the livers with various concentrations of dl-propranolol resulted in a marked reduction in hepatic clearance of lidocaine. Metabolic profile also indicated that hepatic degradation of lidocaine was also significantly inhibited in the dl-propranolol-pretreated livers. Results obtained from the present study and those obtained previously from isolated rat hepatocytes indicate that the observed reduction of hepatic clearance and metabolism of lidocaine by dl-propranolol pretreatment may be the result of binding displacement at the hepatic site.

The pathways of propranolol metabolism in dog and rat liver 10,000g supernatant fractions.

By use of an XAD-2 sample purification procedure, the quantitative patterns of the in vitro metabolism of propranolol and four of its metabolites have been examined by a GC/MS/DATA procedure. Supernatant fractions (10,000g) from both dog and rat livers have been used to degrade propranolol, 4-hydroxypropranolol, N-deisopropylpropranolol, propranolol glycol, and alpha-naphthoxylactic acid. There were both qualitative and quantitative differences in dog and rat metabolism; rat 10,000g supernatant fraction produced more hydroxylation than that of the dog. alpha-Naphthol is not produced from propranolol directly, but rather from alpha-naphthoxylactic acid with an intermediate step requiring NAD+ as a cofactor. We observed no degradation of 4-hydroxypropranolol from the 10,000g supernatant preparations. Thus, 1,4-naphthalenediol is more likely a product of 4-hydroxypropranolol glycol.

Myoinositol uptake by rat hepatocytes in vitro.

Myoinositol uptake by rat hepatocytes in vitro was studied. Adult rat hepatocytes were prepared by digestion of the perfused liver with collagenase. Cell suspensions were incubated with tritium-labeled myoinositol in pH 7.4 Krebs bicarbonate solution containing 1% gelatin at 37 degrees. 14C-Carbon-labeled polyethylene glycol was used as a marker of adherent extracellular fluid volume. Myoinositol uptake was demonstrable after 5 min of incubation, but no intracellular concentration in excess of that in the incubation medium was observed after 60 min of incubation. Uptake saturation over a wide myoinositol concentration range could not be demonstrated. Neither the omission of sodium ions nor the inclusion of ouabain suppressed the distribution ratio significantly. Metabolic inhibitors and lower temperatures also showed no effect. Hexoses, phlorizin or mannitol, exerted no observable effect on myoinositol uptake. The results indicated that myoinositol uptake by rat hepatocytes is probably a passive process.

Quantitative analysis of propranolol and metabolites by a gas chromatograph mass spectrometer computer technique.

A quantitative assay for urinary propranolol and six of its unconjugated metabolites by a gas chromatography mass spectrometry computer system has been developed. Conjugated metabolites are also determined following enzyme hydrolysis. These compounds are separated from murine by Amberlite XAD-2 resin column chromatography and are converted to methyl esters and trifluoroacetyl derivatives. Each substance is quantitated from reconstructed ion chromatograms using a specific mass or sum of masses for each compound by comparing its area with that of an appropriate internal standard. The minimum detectable concentrations are in the range of 50--100 ng ml-1. The calibrations are linear from 0.10--15.0 microgram ml-1 with recoveries ranging from 62--91%. This procedure yields a high degree of specificity, selectivity and reproducibility, as demonstrated by the analyses of urine from dogs receiving single 40 mg doses of propranolol orally.