James W. Bridges

Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment

This review examines the large body of toxicological and epidemiological information on human exposures to chlorpyrifos, with an emphasis on the controversial potential for chlorpyrifos to induce neurodevelopmental effects at low doses. The results of this review demonstrate that the use of urinary 3,5,6-trichlorpyridinol (TCPy), a metabolite of chlorpyrifos as a biomarker of nonoccupational exposure is problematic and may overestimate nonoccupational exposures to chlorpyrifos by 10-to 20-fold because of the widespread presence of both TCPy and chlorpyrifos-methyl in the food supply. Current “background” (nonoccupational) levels of exposure to chlorpyrifos are several orders of magnitude lower than those required to inhibit plasma cholinesterase activity, which is a more sensitive target than nervous system cholinesterase. However, several in vitro studies have identified putative neurodevelopmental mechanisms that are altered at concentrations of chlorpyrifos below those that inhibit cholinesterases. Although one human cohort study reported an association between maternal and cord blood chlorpyrifos levels and several measures of neurodevelopment, two other cohort studies that utilized urinary TCPy as a surrogate for chlorpyrifos exposure did not demonstrate an association. Although the weight of the scientific evidence demonstrates that current levels of chlorpyrifos exposure will not have any adverse effects on neurodevelopment that might result from inhibition of nervous system cholinesterases, several recent studies propose alternative mechanisms. Thus, further in vivo investigation on neurodevelopment in an appropriate animal model is needed; additional epidemiological studies may be warranted if a suitable, chlorpyrifos-exposed cohort can be identified and more rigorous measures of exposure are utilized.

The enzymic isolation of adult rat hepatocytes in a functional and viable state.

Various methods for the preparation of rat hepatocyte suspensions have been compared with regard to cell yield and viability index. A modified method of collagenase/hyaluronidase digestion using a sequential removal and replacement of Ca 2+ and the omission of all perfusion steps has been evolved, thereby making the technique potentially viable for use with pieces of liver such as isolated human liver biopsy material. These suspensions contain large numbers of viable cells, and the viability index is also high (>70%). A number of metabolic functions can be readily detected in these cell suspensions, indicating the intactness of the isolated cells. A systematic survey of the enzymic components required for liver digestion is also reported.

Benzo(a)pyren-3-yl hydrogen sulphate, a major ethyl acetate-extractable metabolite of benzo(a)pyrene in human, hamster and rat lung cultures

Abstract Benzo(a)pyrene is metabolised by human bronchial epithelium to ethyl acetate-extractable metabolites which co-chromatograph with 9,10-dihydro-9,10-dihydroxybenzo(a) pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a) pyrene, whereas little 4,5-dihydroxybenzo(a)pyrene and 3-dydroxybenzo(a)pyrene are formed. Similar results are obtained with human lung except that a major ethyl acetate-soluble metabolite (X) is observed. X has been identified as benzo(a)pyren-3-yl hydrogen sulphate on the basis of enyymic and acid hydrolysis experiments, incorporation of [ 35 S]sulphate and its u.v. and fluorescence spectra which were similar to those of the synthetic metabolite. The u.v. absorption spectrum of benzo(a)pyrene-3-yl hydrogen sulphate is comparable with the X 1 metabolite of benzo(a)pyrene, one of the principal metabolites of unestablished identity found in earlier in vivo studies. The biological activity of the sulphate ester of 3-hydroxybenzo(a)pyrene is of interest as this metabolite could be extremely persistent in man because of its physico-chemical properties, which may prevent its excretion in the urine and bile.

Time and dose-response study of the effects on rats of the plasticizer di(2-ethylhexyl) phthalate

Groups of male and groups of female Wistar albino rats were administered diets containing sufficient di(2-ethylhexyl) phthalate (DEHP) to ensure intakes of either 1000, 200, or 50 mg/kg/day. Four rats from each experimental group and six control rats of the same sex were killed 3, 7, 14, and 28 days and 9 months after commencement of treatment. At all time points the major abdominal organs were removed and subjected to histological examination. A more extensive necropsy was performed on those rats killed after 9 months of treatment. At all time points the livers of the rats were subjected to extensive histologic, electron microscopic, and biochemical examination. Changes could be grouped according to their time course. Two early and transient alterations were noticed. First, there were morphologic changes in the bile canaliculi of male rats treated with 1000 mg/kg/day of DEHP. Second, there was a burst of mitosis immediately after the start of administration of the compound. The time course of this mitotic burst varied; the increase in mitosis was greatest at 3 days in rats treated with 1000 mg/kg/day of DEHP and was smaller but more prolonged in rats treated with 200 or 50 mg/kg/day. Other changes, namely, a midzonal to periportal accumulation of fat, induction of peroxisomal enzymes, and induction of the P-450 isoenzyme also developed rapidly but were sustained throughout the study. The maximal change was usually attained within 7 days of commencement of treatment. More slowly developing changes were hypertrophy of the hepatocytes, centrilobular loss of glycogen, and a fall in glucose-6-phosphatase activity. Here maximal changes were not attained until 28 days after commencement of treatment. These three effects were clearly observed in rats treated with 200 or 1000 mg/kg/day of DEHP but were only marginally altered in rats treated with 50 mg/kg/day. Finally accumulation of lipid-loaded lysosomes assessed by light and electron microscopy and by assay of beta-galactosidase activity was only apparent in rats treated with DEHP for 9 months with 200 or 1000 mg/kg/day of DEHP. Changes in female rats were qualitatively similar to those observed in male rats. The alterations were, however, less pronounced than in male rats treated with an equal dose of DEHP and the degree of liver enlargement was much less because, although the initial hyperplasia was clearly apparent, there was a much smaller degree of hypertrophy.

Factors influencing peroxisome proliferation in cultured rat hepatocytes.

A primary rat hepatocyte culture system has been developed for the study of peroxisome proliferation. Maximal induction of peroxisomal activity requires supplementation of the culture medium with hydrocortisone. The addition of clofibric acid (0.01–1 mM), mono-(2-ethylhexyl)phthalate (0.01–0.5 mM) and trichloroacetic acid (0.1–5 mM) to cultured rat hepatocytes resulted in a time- and dose-related increase in CN- insensitive palmitoyl CoA oxidation (maximal increases: 27-, 15.5-, and 5-fold respectively) and mitochondrial α-glycerophosphate dehydrogenase activity (maximal increases: 7.3-, 5.8-, and 1.6-fold respectively). Electron microscopic examination revealed smooth endoplasmic reticulum proliferation and morphometric analysis indicated an increase in fractional peroxisomal volume of X 8 and X 4 for clofibric acid (1 mM) and trichloroacetic acid (2.5 mM), respectively. SDS-PAGE of cell homogenates revealed an intensified protein band of mol. wt. 76–78,000. The induction of peroxisomal β-oxidation by clofibric acid was elevated from 9- to 12-fold by supplementation of the medium with l-carnitine (2mM).

Comparison of the short-term effects of di(2-ethylhexyl) phthalate, di(n-hexyl) phthalate, and di(n-octyl) phthalate in rats

This study compares changes in the livers of rats treated with di(2-ethylhexyl) phthalate (DEHP) and its straight-chain analogs di(n-hexyl) phthalate (DnHP) and di(n-octyl phthalate (DnOP). Groups of rats were fed diets containing 20,000 ppm of one of these compounds. Subgroups were killed after 3, 10, and 21 days, and the livers were examined by histological, cytological, and biochemical methods. The results show considerable differences between the effects of the branched-chain phthalate ester DEHP and its straight-chain analogs. The major effects on the liver following administration of diets containing DEHP were midzonal and periportal accumulation of small droplets of lipid, hepatomegaly accompanied by an initial burst of mitosis, proliferation of hepatic peroxisomes and of smooth endoplasmic reticulum accompanied by induction of peroxisomal fatty acid oxidation, damage to the peroxisomal membranes as evidenced by increased leakage of catalase to the cytosol, and centrilobular loss of glycogen and falls in glucose-6-phosphatase activity and in low-molecular-weight reducing agents. In contrast, diets containing DnHP or DnOP induced accumulation of large droplets of fat around central veins leading, by 10 days, to mild centrilobular necrosis and a very slight induction of one peroxisomal enzyme and an increase in liver weight, but no significant changes in any other parameters which were affected by DEHP.

Chemically Induced Renal Papillary Necrosis and Upper Urothelial Carcinoma. Part 1

In the past, renal papillary necrosis (RPN) has been commonly associated with long-term abusive analgesic intake, but over recent years a wide variety of industrially and therapeutically used chemicals have been shown to induce this lesion experimentally or in man. Destruction of the renal papilla may result in: (1) secondary degenerative cortical changes which precede chronic renal failure or (2) a rapidly metastasizing upper urothelial carcinoma, which has a very poor prognosis. This article will briefly review the published data on the morphology, function, and biochemistry of the normal renal medulla and the pathology associated with RPN, together with the secondary changes which give rise to cortical degeneration or epithelial carcinoma. It will then examine in detail those chemicals which have been reported to cause RPN in an attempt to delineate structure-activity relationships. Finally, the many different theories that have been proposed to explain the pathophysiology of RPN will be examined and an hypothesis will be put forward to explain the primary pathogenesis of the lesion and its secondary consequences.

Studies on the metabolism and excretion of Benzo(a)pyrene in isolated adult rat hepatocytes

Abstract [ 3 H]Benzo(a)pyrene is metabolised by isolated rat hepatocytes to both ethyl acetate-soluble metabolites, which co-chromatograph with 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene and its sulphate ester, benzo(a)pyren-3-yl-hydrogen sulphate, and to water-soluble metabolites. Hydrolysis of the water-soluble metabolites with β-glucuronidase release ethyl acetate-soluble metabolites which co-chromatograph with 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 4,5-dihydro-4,5-dihydrobenzo(a)pyrene, 9,10-dihydroxybenzo(a)pyrene (9,10-catechol) and 3-hydroxybenzo(a)pyrene. During the incubation significant differences in the distribution of metabolites between the cells and the extracellular medium are observed. Initially the cells produce predominantly ethyl acetate-soluble metabolites, which are only partly released into the extracellular medium, but at later times in the incubation a greater percentage of the metabolites are further metabolised to water-soluble conjugates which are very readily released from the cells. Individual ethyl acetate-soluble metabolites show significant distributional differences. Monohydroxybenzo(a)pyrenes accumulates intracellularly and only low amounts are released into the medium. Sulphate esters of monohydroxybenzo(a)pyrenes such as benzo(a)pyren-3-yl-hydrogen sulphate also accumulate intracellularly, although to a lesser extent than the monohydroxybenzo(a)pyrenes. 4,5-Dihydro-4,5-dihydroxybenzo(a)pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene are distributed more evenly between cells and medium whereas 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene is found mainly in the medium. Significant amounts of radioactivity are bound irreversibly to cellular macromolecules.

Enzyme immunoassay: a review.

In the last few years, the use of enzyme labels in immunoassays has been investigated. The aim of this review is to evaluate critically the role of such labels in clinical biochemistry. Special attention has been given to the problems involved in preparing enzyme labels and the ways in which such labels can be used in a variety of heterogeneous and homogeneous assay systems.

Studies on the interaction of safrole with rat hepatic microsomes

Abstract (1) Similar to previous results with methylenedioxyphenyl compounds microsomes from safrole pretreated rats showed, on reduction with NADH, NADPH or Na 2 S 2 O 4 , characteristic absorption maxima at 427 and 455 nm. The same spectrum can be obtained after incubation in vitro of control microsomes with safrole, NADPH and oxygen. (2) Subsequent addition of carbon monoxide to microsomes of safrole pretreated rats causes an absorption maximum at 448 nm, characteristic of the 3-methylcholanthrene type of induction of microsomal hydroxylase protein. (3) The suspected cytochrome P-450-safrole metabolite complex, which can be visualized only in the reduced state of cytochrome P-450, is very stable as witnessed by its preservation through the preparation procedure for microsomes or after dialysis or detergent treatment. However, when safrole or ethylbenzene is added, both absorption maxima decrease in a time dependent manner. This can be measured for each time point after complete reduction of the microsomal preparation by adding Na 2 S 2 O 4 . (4) From this it is concluded that the carcinogen safrole leads to the biosynthesis of a 3-methylcholanthrene type cytochrome P-450 and formation of a safrole metabolite-cytochrome P-450 complex which in turn can be cleaved in vitro by safrole or other lipophilic compounds.