Flow cytometry, morphometry and histopathology as biomarkers of benzo[a]pyrene exposure in brown bullheads (Ameiurus nebulosus).

Brown bullheads were given a single intraperitoneal dose of 0, 5, 25 or 125 mg kg-1 benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon, and evaluated over 18 months. Flow cytometric analyses of hepatocyte DNA content indicated an increase in DNA synthesis in BaP-exposed fish prior to day 14 post-exposure. Thereafter, all flow cytometric variables returned to initial levels. Histopathological evaluation of livers from fish sampled at 18 months revealed significant differences among treatments in the amount of hepatic macrophage ceroid pigmentation and basophilic staining intensity. No neoplasms or changes in blood cell DNA content were detected. Significant morphometric variations existed among fish, but differences between sexes overshadowed differences attributable to dose. Flow cytometry yielded no evidence of long-term DNA alterations from a single exposure to BaP; however, the differences detected by DNA analysis shortly after the toxic event suggest that flow cytometric cell cycle analysis may be useful for documenting continuing exposures.

Microcontaminants and reproductive impairment of the Forster's tern on Green Bay, Lake Michigan--1983.

For the 1983 nesting season, Forster's tern (Sterna forsteri) reproductive success was significantly impaired on organochlorine contaminated Green Bay, Lake Michigan compared to a relatively uncontaminated inland location at Lake Poygan, Wisconsin. Compared with tern eggs from Lake Poygan, eggs from Green Bay had significantly higher median concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), other polychlorinated dibenzo-p-dioxins (PCDDs), total polychlorinated biphenyls (PCBs), total (three congeners) non-ortho, ortho' PCBs, five individual PCB congeners known to induce aryl hydrocarbon hydroxylase (AHH) and several other organochlorine contaminants. Conversions of analytical concentrations of TCDD and PCB congeners based on relative AHH induction potencies allowed for estimation of total 2,3,7,8-TCDD equivalents. Two PCB congeners, 2,3,3',4,4'- and 3,3',4,4',5-pentachlorobiphenyl (PeCB) accounted for more than 90% of the median estimated TCDD equivalents at both Green Bay and Lake Poygan. The median estimated TCDD equivalents were almost 11-fold higher in tern eggs from Green Bay than in eggs from Lake Poygan (2175 and 201 pg/g). The hatching success of Green Bay sibling eggs from nests where eggs were collected for contaminant analyses was 75% lower at Green Bay than at Lake Poygan. Hatchability of eggs taken from other nests and artificially incubated was about 50% lower for Green Bay than for Lake Poygan. Among hatchlings from laboratory incubation, those from Green Bay weighed approximately 20% less and had a mean liver weight to body weight ratio 26% greater than those from Lake Poygan. In both field and laboratory, mean minimum incubation periods were significantly longer for eggs from Green Bay compared to Lake Poygan (8.25 and 4.58 days, respectively). Mean minimum incubation time for Green Bay eggs in the field was 4.37 days longer than in the laboratory. Hatchability was greatly improved when Green Bay eggs were incubated by Lake Poygan adults in an egg-exchange experiment, but was sharply decreased in Lake Poygan eggs incubated in Green Bay nests. Nest abandonment and egg disappearance were substantial at Green Bay but nil at Lake Poygan. Thus, not only factors intrinsic to the egg, but also extrinsic factors (parental attentiveness), impaired reproductive outcome at Green Bay. The epidemiological evidence from this study strongly suggested that contaminants were a causal factor. AHH-active PCB congeners (intrinsic effects) and PCBs in general (extrinsic effects) appeared to be the only contaminants at the concentrations measured in eggs, capable of producing the effects that were observed at Green Bay.

Classification of lung cancer patients and controls by chromatography of modified nucleosides in serum.

A wide spectrum of modified nucleosides has been quantified by high-performance liquid chromatography in serum of 49 male lung cancer patients, 35 patients with other cancers, and 48 patients hospitalized for nonneoplastic diseases. Data for 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis and stepwise discriminant analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer and 75% of the noncancer subjects. It also demonstrated 84% sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity in comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum and show that precise measurement of an array of 29 modified nucleosides in serum by high-performance liquid chromatography with UV scanning with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring.

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver.

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver phenobarbital which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP), alpha-Naphthoflavone (ANF) selectivity inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (Mr = 56,000). However, HCB did induce a polypeptide with Mr = 53,000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the 'mixed-type' induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.

Negative chemical ionization studied of human and food chain contamination with xenobiotic chemicals.

Negative chemical ionization mass spectrometry with a mixture of isobutane, methylene chloride, and oxygen as the reagent gas has been used to explore contamination of environmental substrates with xenobiotic chemicals. The substrates in question, fish tissue, human seminal plasma, and human adipose tissue, were cleaned up by one of the following three cleanup procedures: (1) continuous liquid-liquid extraction steam distillation; (2) gel-permeation chromatography; and (3) adsorption on activated carbon followed by elution with toluene. The third procedure was used only for the examination of planar polychlorinated aromatic hydrocarbons in environmental samples. Using these techniques, we have found evidence for contamination of fish samples with polychloronaphthalenes, polychlorostyrenes, polychlorobiphenyls, polychlorodibenzofurans, and polychlorodibenzodioxins among other chemicals. The polychlorodibenzodioxins appeared only in the spectra of extracts of fish obtained from the Tittabawassee River at Midland Michigan. The polychlorodibenzofuran ions appeared in NCI mass spectra of fish that were significantly contaminated (above 2 ppm) with polychlorobiphenyls. Toxic substances occurring in human seminal plasma included pentachlorophenol, hexachlorobenzene, DDT metabolites, and polychlorobiphenyls. We have investigated toxic substances in human seminal plasma because of the apparent decrease in sperm density in U.S. males over the last 30 years. Results of screening human adipose tissue for contamination with xenobiotic chemicals have been largely coincident with result of the EPA human monitoring program. Polychlorobiphenyls, DDT metabolites, nonachlor, and chlordane have appeared in most samples examined. Detection limits for all of these chemicals were of the order of 1 ppb.

Silicic acid chromatographic separation of polychlorinated biphenyls and pesticides: some contaminants and limitations.

The Armour and Burke method for separating polychlorinated biphenyls (PCBs) from pesticides involves the use of silicic acid. However, we detected di-2-ethylhexyl phthalate, PCBs, and other impurities as interfering contaminants in several batches of silicic acid. The presence of H2SO4 in some batches of silicic acid is inferred. The acid may be responsible for the production of contaminants which interfere in gas-liquid chromatographic analyses. Contaminants in silicic acid are reduced by extracting the adsorbent with solvent, and/or partitioning the concentrated pesticide fraction with 1N NaOH. These purification procedures provide separations relatively free of impurities, but result in reduced adsorbent activity. PCB-pesticide separations are reproducible only within a given batch lot of silicic acid because of the varying adsorbent characteristics of each lot. Alternative adsorbents should be explored for most PCB-pesticide separations.