Cell proliferation and chemical carcinogenesis: a symposium overview.
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Animals↗
Biomedical subjects
G Lucier
Publications and source records attributed to G Lucier.
Ligand/receptor binding for 2,3,7,8-TCDD: implications for risk assessment.
There is renewed controversy regarding safe exposure levels for dioxin. At the heart of this controversy is the hypothesis that toxic effects of dioxin are receptor-mediated and therefore a "threshold" should exist below which no toxic effects can occur. Our research focuses on the ability of dioxin to alter protein levels in rodent livers. Established effects of exposure to dioxin are the induction of cytochrome P450-1A1 and P450-1A2 and a reduction in the maximal binding of the epidermal growth factor receptor in rat livers. An initiation-promotion protocol was used to study the effects of dioxin in female Sprague-Dawley rats. Animals were administered a single initiating dose of diethylnitrosamine followed by 16 biweekly gavage doses of 2,3,7,8-TCDD. Steady-state pharmacodynamic models were fit to these data assuming a combination of Hill kinetics and Michaelis-Menten kinetics. Two classes of models were developed which postulate two different mechanisms for the constitutive expression and TCDD-induced alterations in the levels of these proteins. The results are consistent with models which follow proportionate response in the low-dose region (no threshold) and with models which allow for a low-dose threshold. In all cases studied, the best fitting model exhibited no "threshold" for the effects of TCDD on the modulation of these proteins. The finding is consistent with the knowledge that for some receptor-mediated responses, there is a proportional relationship between receptor occupancy and biological response, even at low ligand concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Environmental toxicology of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans.
Few environmental compounds have generated as much interest and controversy within the scientific community and in the lay public as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Their ubiquitous presence in the environment and the risk of accidental exposure has raised concern over a possible threat of PCDDs or PCDFs to human health. The most extensively studied and potent isomer is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). Dioxin is a multisite toxicant in laboratory rodents resulting in a number of tissue-, species-, and sex-dependent responses. Much has been learned about the mechanism of dioxin's effects, especially for the induction of cytochrome P-450 enzymes. Binding of PCDDs and PCDFs to a receptor protein, termed the dioxin or Ah receptor, is necessary for most biological and toxic responses. The most common toxic response used for evaluating the human health risk posed by PCDDs and PCDFs is the hepatocarcinogenic response observed primarily in rodents. Despite extensive research efforts, the effects of PCDDs and PCDFs on humans are not well characterized. However, available data indicate there is good agreement between known effects of dioxin in laboratory animals and those described in epidemiological studies for effects in humans. The sequence in events initiated by the Ah receptor interacting with dioxin-responsive genes and ending with altered patterns of differentiation and growth must be sought in order to understand tissue, species, sex, and interindividual variation in biological responses and the health risk posed by PCDDs and PCDFs.
Animals↗
Dose response for TCDD promotion of hepatocarcinogenesis in rats initiated with DEN: histologic, biochemical, and cell proliferation endpoints.
The present study examines the dose-response relationship for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promotion of histologic and biochemical parameters by using a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats initiated with a single intraperitoneal dose of 175 mg of diethylnitrosamine (DEN)/kg body weight at 70 days of age. Starting 2 weeks after initiation, treatment groups of 8-10 rats were given TCDD by gavage in corn oil once every 2 weeks for 30 weeks. Doses were 3.5, 10.7, 35.7, and 125 ng TCDD/kg body weight/day. A significant body weight reduction was present in the noninitiated group that received 125 ng TCDD. Relative liver weight was statistically increased in initiated rats treated with > or = 10.7 ng TCDD and in noninitiated rats treated with > or = 35.7 ng TCDD. Histopathologic evidence of cytotoxicity was dose-related in all TCDD-treated groups. There was a statistically significant dose response in the bromodeoxyuridine (BrdU) S-phase labeling index (LI) in the DEN-initiated rats (p < 0.01) and a marginally significant trend in the saline-treated rats (p = 0.10), but proliferating cell nuclear antigen S-phase LI and growth fraction within altered hepatic foci showed no increase. Among the DEN-initiated groups there was a significant increase in glutathione S-transferase altered hepatic foci stereological parameters in the 125 ng TCDD group. This study demonstrates that dose-response relationships for TCDD's effects on cell proliferation growth of altered hepatic foci are different from previously reported effects on P450 gene expression, indicating that different biological or biochemical responses may exhibit different dose-response relationships.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cell proliferation and chemical carcinogenesis: symposium overview.
Cancer, by definition, is a proliferative disease. The fundamental scientific issue explored at the international symposium "Cell Proliferation and Chemical Carcinogenesis" was the impact of chemically enhanced cell proliferation on the dynamic carcinogenic processes. This conference, held at the National Institute of Environmental Health Sciences January 14-16, 1992, provided an open forum for the exchange of new results, information, and ideas in four areas: a) general principles of cell division and carcinogenesis, b) critical evaluation of cell proliferation methodologies, c) cell proliferation and modeling of organ-specific carcinogenesis, and d) cell proliferation and human carcinogenesis. This overview summarizes key findings from that symposium. The general view expressed was that although cell proliferation is involved inextricably in the development of cancers, chemically enhanced cell division does not reliably predict carcinogenicity. Our knowledge of the multistep nature of carcinogenesis has advanced substantially during recent years; however, much still needs to be learned. A greater understanding of the cellular and molecular events in chemical carcinogenesis should improve all aspects of the overall risk assessment process, including extrapolations based on dose, species, and interindividual differences.
Animals↗
Perspectives on risk assessment impact of recent reports on benzene.
Very sensitive methods that can detect the benzene metabolite muconic acid (MA) in the urine of virtually all members of the general population have recently become available and have been used in a few occupational studies as a marker of benzene exposure. Preliminary findings from these studies suggest that urinary MA may be a reliable marker of occupational exposure to greater than 5 ppm benzene. It was also consistently observed that a certain proportion of the general population have urinary MA levels compatible with those seen in persons occupationally exposed to greater than 1 ppm benzene. It is unlikely that these elevated levels can be explained solely as being artifactual. The frequency with which they occur for a given individual, and the duration with which they are maintained, are not known. Information on these two factors is needed in order to adequately assess whether or not these levels present a significant risk for a segment of the general population.
Benzene↗
S-phenylcysteine formation in hemoglobin as a biological exposure index to benzene.
Benzene is metabolized to intermediates that bind to hemoglobin, forming adducts. These hemoglobin adducts may be usable as biomarkers of exposure. In this paper, we describe the development of a gas chromatography/mass spectroscopy assay for quantitating the binding of the benzene metabolite, benzene oxide, to cysteine groups in hemoglobin. We used this assay to study the hemoglobin adduct, S-phenylcysteine (SPC), in the blood of rats and mice exposed to benzene either by inhalation or by gavage. We were able to detect SPC in the hemoglobin of exposed rats and mice, to show the linearity of the exposure dose-response relationship, and to establish the sensitivity limits of this assay. For the same exposure regime, rats showed considerably higher levels of SPC than did mice. As yet, we have not been able to detect SPC in the globin of humans occupationally exposed to benzene. We attempted to determine whether the SPC found in hemoglobin originated from the metabolism of benzene within or outside of the red blood cell. We hypothesized that the greatest red blood cell metabolism would be associated with peripheral reticulocytes, which retain high metabolic capacity. After exposing rats to benzene, we isolated the red blood cells and used discontinuous Percoll gradients to fractionate them into age groups. No differences in SPC levels were found among any of the fractions, suggesting that the SPC found in globin originates from the metabolism of benzene to benzene oxide in a location external to the red blood cell. To our knowledge, this is the first demonstration of the nonenzymatic binding of the benzene metabolite, benzene oxide, to protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Metabolism of [14C]benzene by cynomolgus monkeys and chimpanzees.
Rodent bioassays indicate that B6C3F1 mice are more sensitive to the carcinogenicity of benzene than are rats. The urinary profile of benzene metabolites is different in rats vs mice. Mice produce higher proportions of hydroquinone conjugates and muconic acid, indicators of metabolism via pathways leading to putative toxic metabolites, than do rats. In both species, metabolism to hydroquinone and muconic acid is favored at low concentrations of benzene, indicating that these pathways are easily saturated. These species differences in the metabolism of benzene make it difficult to predict the health risk to humans and how this risk varies with dose. For this reason, the metabolism of [14C]benzene by cynomolgus monkeys and chimpanzees, animals phylogenetically closer to humans than rodents, was studied. Monkeys were dosed ip with 5, 50, or 500 mg [14C]benzene/kg body wt. Urine was collected for up to 24 hr following exposure and was analyzed for benzene metabolites. The proportion of the administered 14C excreted in the urine of monkeys decreased from approximately 50 to 15% as the dose increased. Phenyl sulfate was the major urinary metabolite. The proportion of hydroquinone conjugates and muconic acid in the monkey's urine decreased as the dose increased. The proportion of catechol conjugates was not affected by dose. The proportion of these metabolites in the urine was quite variable from animal to animal, but the proportion of muconic acid was consistently much lower in the monkey than in the mouse or rat. Three chimpanzees were administered 1 mg [14C]benzene/kg body wt, iv; essentially all of the injected 14C was recovered in the urine. Of the total urinary metabolites, 79% were accounted for by phenyl conjugates and less than 15% by hydroquinone conjugates or muconic acid. Catechol conjugates were not detected. The metabolism of benzene appeared to be qualitatively similar but quantitatively different in the species studied. The mouse, the sensitive rodent species, forms the highest levels of hydroquinone conjugates and muconic acid and the chimpanzee, the lowest. In all animal species studied for the effect of dose on benzene metabolism, as the dose decreased, a larger proportion of the benzene metabolites was represented by hydroquinone conjugates and muconic acid.
Animals↗
Integrated approach for evaluating species and interindividual differences in responsiveness to dioxins and structural analogs.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that is produced inadvertently during the synthesis of some organochlorine compounds, such as the chlorinated phenoxy pesticides. It is biologically and ecologically persistent, with an estimated half-life of 7 years in humans. It possesses high acute toxicity in rodents and is a carcinogen, teratogen, and immunotoxin. In chronic bioassays for carcinogenicity, TCDD at a dose of 10 ng/kg/day increases the incidence of liver tumors in female rats, making it one of the most potent animal carcinogens ever tested. A recent study in humans has shown an increase in the incidence of respiratory tract tumors in workers in chlorinated phenoxy herbicide plants. Considerable controversy and uncertainty remain, however, concerning its carcinogenic potency in humans and the reliability of using animal data to predict human risks. It is generally accepted that most, if not all, of the effects of TCDD require its binding to the Ah receptor. In addition to its toxic effects, TCDD produces a number of biochemical effects, such as induction of CYP1A1, downregulation of binding activity of the estrogen and epidermal growth factor (EGF) receptors, and changes in cytokine pathways. These effects suggest that the Ah receptor plays an important role in regulating the cell cycle. A number of structural analogs of TCDD, such as the polychlorinated dibenzofurans, also interact with the Ah receptor, and they produce the same spectrum of responses as TCDD in animal and cell models. The potency of these compounds is strongly correlated with their binding affinity to the Ah receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Muconic acid determinations in urine as a biological exposure index for workers occupationally exposed to benzene.
Urinary phenol determinations have traditionally been used to monitor high levels of occupational benzene exposure, but the same technique cannot be used to monitor low-level exposures because of the high background of phenol resulting from its presence in many foods and from metabolism of aromatic amino acids. Thus, new biological indexes for exposure to low levels of benzene are needed. Animal studies indicate that muconic acid is a metabolite of benzene that is excreted in the urine as an increasing fraction of the total benzene metabolites with decreasing dose of benzene. Thus, urinary muconic acid is potentially useful as a monitor for low levels of exposure to benzene. It is also of interest to determine the level of muconic acid in the urine of humans exposed to benzene for comparison with animal data as an aid for use of the animal studies in risk assessments for humans. This report describes the development of a gas chromatography/mass spectrometry assay to detect and quantitate the benzene metabolite, muconic acid, in urine. The internal standard used in the assay, muconic acid-d4, was biosynthesized by F344/N rats administered benzene-d6 by gavage; the muconic acid was isolated from the rat's urine. Muconic acid was measured in experimental urine samples by adding the internal standard, followed by extraction and derivatization. Phenol was also measured in urine after extraction and derivatization. The assays were applied to the urine samples from 14 workers occupationally exposed to benzene and 8 workers with no known benzene exposure. Muconic acid could be detected in all of the urine samples at levels greater than 100 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei.
Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344/N rats and B6C3F1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased. Exposure to the same level of benzene for an additional 2 weeks did not further increase the frequency of micronuclei in PCEs. These results indicate that repeated exposures to benzene, such as might be encountered by humans as a result of occupational or environmental exposures, are not likely to change or increase benzene metabolism.
Administration, Inhalation↗
Benzene hemoglobin adducts in mice and rats: characterization of formation and physiological modeling.
Benzene is a myelotoxin and a human leukemogen. Humans are exposed to this compound, both occupationally and environmentally. This study was conducted to determine whether formation of benzene-derived adducts with blood hemoglobin (Hb) can be used as a biomarker of exposure to benzene. B6C3F1 mice and F344/N rats were given 0.1 to 10,000 mumol [14C]benzene/kg body wt, orally. Twenty-four hours later, animals were euthanized, and globin was isolated from blood samples. The globin was analyzed by liquid scintillation spectrometry for the presence of [14C]benzene-derived adducts. Hb adduct formation was linear with respect to dose for amounts of up to 500 mumol [14C]benzene/kg body wt, for both rodent species. Within this linear dose-response range, mice formed adducts from [14C]benzene approximately 3.5 times less efficiently [0.022 +/- 0.010 (pmol adducts/mg globin)/(mumol/kg body wt dose)] than did rats [0.076 +/- 0.014 (pmol adducts)/(mumol/kg body wt dose)]. Benzene-derived Hb adducts also accumulated linearly when mice and rats were given up to three daily doses of 500 mumol [14C]benzene/kg body wt. These data were used to develop a physiological model for benzene-derived Hb adduct formation. Both first-order and saturable pathways for adduct formation were incorporated. The results showed that the model simulated the levels of Hb adducts in both mice and rats after oral exposures to benzene and predicted the levels of Hb adducts present after inhalation exposure. These studies suggest that Hb adducts might be useful biomarkers for human exposures to benzene.
Animals↗
Dose-response relationships in promotion of rat hepatocarcinogenesis by 17 alpha-ethinylestradiol.
One of the critical issues in risk assessment for chemical carcinogens is the evaluation of dose-response relationships for tumor promoters. In the studies reported here we have systematically investigated dose-response relationships for the liver tumor-promoting actions of 17 alpha-Ethinylestradiol (EE2) following a single injection of diethylnitrosamine (200 mg/kg) to ovariectomized female rats. Parameters measured included tumor incidence, gamma-glutamyltranspeptidase (GGT) positive foci, serum prolactin and serum EE2. The length of tumor promotion ranged from 30 to 60 wk. Results showed a linear increase in GGT-positive foci between doses of 16 and 90 micrograms EE2 kg/d for 30 wk. This was associated with corresponding increases in liver tumor incidence at 60 wk. Seventy-five percent of the animals had either hepatocellular adenoma or hepatocellular carcinoma in the group promoted with 90 micrograms EE2/kg for 60 wk. No liver tumors were evident in either controls or animals receiving estrogen only. Serum prolactin concentrations were elevated in all estrogen-treated groups. In summary, our studies have evaluated dose-response relationships for GGT-positive foci and tumor incidence in a two-stage model for hepatocarcinogenesis using EE2 as the promoting agent.
Animals↗
The effects of TCDD on receptors for epidermal growth factor, glucocorticoid, and estrogen in Ah-responsive and -nonresponsive congenic mice and the effects of TCDD on estradiol metabolism in a liver tumor promotion model in female rats.
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Animals↗
A physiological model for simulation of benzene metabolism by rats and mice.
Studies conducted by the National Toxicology Program on the chronic toxicity of benzene indicated that B6C3F1 mice are more sensitive to the toxic effects of benzene than are F344 rats. A physiological model was developed to describe the uptake and metabolism of benzene in rats and mice and to determine if the observed differences in toxic effects could be explained by differences in the pathways for metabolism of benzene or by differences in uptake of benzene. Major pathways for elimination of benzene included metabolism to hydroquinone glucuronide or hydroquinone sulfate, phenyl glucuronide or phenyl sulfate, muconic acid, and prephenyl mercapturic acid or phenyl mercapturic acid. Model simulations for total benzene metabolized and for profiles of benzene metabolites were conducted for oral or inhalation exposure and compared to data for urinary excretion of benzene metabolites after exposure of rats and mice to [14C]- or [3H]-benzene by inhalation or gavage. Results for total amount of benzene metabolized, expressed per kilogram body weight, indicated that for inhalation exposure concentrations up to 1000 ppm, mice metabolized at least two to three times as much benzene as did rats. Simulations of oral exposure to benzene resulted in more benzene metabolized per kilogram body weight by rats at oral exposures of greater than 50 mg/kg. Patterns of metabolites formed after either route of exposure were very different for F344/N rats and B6C3F1 mice. Rats primarily formed the detoxification metabolite, phenyl sulfate. Mice formed hydroquinone glucuronide and muconic acid in addition to phenyl sulfate. Hydroquinone and muconic acid are associated with pathways leading to the formation of the putative toxic metabolites of benzene. Metabolic rate parameters, Vmax and Km, were very different for hydroquinone conjugate and muconic acid formation compared to formation of phenyl conjugates and phenyl mercapturic acids. Putative toxication pathways could be characterized as high affinity, low capacity whereas detoxification pathways were low affinity, high capacity. Model simulations suggested that for both rats and mice at lower exposure concentrations hydroquinone and muconic acid represented a larger fraction of the total benzene metabolized than at higher exposure concentrations where detoxification metabolites were predominant. Preferential production of a putative toxic metabolite at low exposure concentrations may have important implications in risk assessment for benzene.
Administration, Inhalation↗
Effect of exposure concentration, exposure rate, and route of administration on metabolism of benzene by F344 rats and B6C3F1 mice.
To determine the effect of exposure concentration and the route of administration on benzene metabolism, male F344/N rats and B6C3F1 mice were orally exposed to 1, 10, and 200 mg benzene/kg, and by inhalation for 6 hr to 5, 50, and 600 ppm benzene vapor. The effect of different exposure rates on the metabolism of benzene was determined by exposing rodents over different time intervals to the same total amount of benzene [constant concentration X time factor (C X T) = 300 ppm.hr]. Water-soluble metabolites constituted greater than 90% of the metabolite dose to the tissues and were used as a measure of the metabolism of benzene via different pathways. Water-soluble metabolites were measured in the blood, urine, liver, lung, and bone marrow from animals killed following oral exposures and during and following inhalation exposures. The total "dose" to the tissue of individual metabolites was determined by the area under the curve (AUC). The results indicated a shift in metabolism from putative toxification pathways to detoxification pathways as the exposure concentration or oral dose increased. In mice, hydroquinone glucuronide and muconic acid (markers of toxification metabolic pathways) represented a greater percentage of the administered dose at low doses than at high doses. At high doses, phenylglucuronide and prephenylmercapturic acid (detoxification products) increased as a percentage of the administered dose. This same metabolic shift was observed in rats, except that hydroquinone glucuronide was a minor metabolite of benzene at all concentrations. The AUC of phenylsulfate (detoxification pathway) was proportional to the exposure concentration in both species. Within the range of C X T factors studied, the rate of the inhalation exposure to benzene did not affect the AUC of metabolites in tissues of rats; however, a high dose rate (600 ppm 0.5 hr) in mice caused a shift in metabolism to phenyl conjugates. The comparison of oral and 6-hr inhalation exposures indicated that, in terms of metabolite dose to tissues, there is no simple relationship between these two routes of administration. An oral dose and an inhalation exposure concentration which produce an equal dose of one metabolite produce very different doses of another metabolite. These studies demonstrated a species difference in benzene metabolism, as well as a metabolic shift in benzene metabolic pathways as the exposure concentration was increased.(ABSTRACT TRUNCATED AT 400 WORDS)
Administration, Inhalation↗
A toxikinetic model for simulation of benzene metabolism.
People exposed to benzene, an important industrial solvent and a common pollutant, can develop aplastic anemia and leukemia. The objectives of this study were to develop a physiological model for the metabolism of benzene, based on studies in laboratory animals, and to use this model to predict benzene metabolism in people to concentrations near the current permissible exposure limits. Model simulations predicted that for 8-h inhalation exposures to below 10 ppm, hydroquinone metabolites would predominate. Hydroquinone is associated with pathways leading to the formation of the putative toxic metabolite, benzoquinone. Lower levels of muconic acid, a marker for the putative toxic metabolite, muconaldehyde, were predicted. At concentrations above 10 ppm, detoxification metabolites such as the phenyl conjugates predominate. Predictions of benzene metabolism in humans based on our physiological model may have important implications for risk assessment. Because there may be preferential production of a putative toxic metabolite at low exposure concentrations, linear extrapolation of toxicity observed at high concentrations may underestimate risk at low exposure concentrations.
Animals↗
Effect of repeated benzene inhalation exposures on subsequent metabolism of benzene.
Benzene is a known human leukemogen and animal carcinogen. To better assess the risks associated with benzene exposure, it would be helpful to determine whether repeated inhalation exposures would affect the metabolism of benzene. The purpose of these experiments was to determine if exposure of F344 rats and B6C3F1 mice to 600 ppm benzene, 6 h/day, 5 days/week for 3 weeks, would affect the subsequent in vivo metabolism of inhaled [14C]benzene.
Administration, Inhalation↗