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T J Gray

Publications and source records attributed to T J Gray.

At least 55 records · Page 3Linked to original sources
PubMed1987

Peroxisome induction studies on seven phthalate esters.

Seven phthalate esters, representing a variety of chain lengths and degrees of branching in the alcohol moiety, were tested for their ability to produce peroxisome proliferation in the Fischer 344 rat. Di(2-ethylhexyl)adipate (DEHA) was tested using the same protocol and di(2-ethylhexyl)phthalate (DEHP) was run with each study as an internal control. Each ester was administered in the feed for a period of 21 days at levels of 2.5%, 1.2% and either 0.6% or 0.3%. DEHP and DEHA were also fed at levels of 0.1% and 0.01%. The animals were sacrificed and samples of liver were prepared for both light and electron microscopy. Serum samples were assayed for both triglyceride and cholesterol. The remaining portion of the liver was homogenized and assayed for cyanide-insensitive palmitoyl-CoA oxidation, lauric acid 11-hydroxylase and lauric acid 12-hydroxylase. The results show that there is approximately a ten-fold difference between the weakest and strongest esters in terms of their potency to induce changes in relative liver weight and in several of the biochemical parameters. In general, the longer chain esters were more potent than the shorter chain ones, and branched chain esters seemed more potent than straight. Several statistical analyses of the dataset have been performed and all render similar conclusions. The results of one of these evaluations are presented elsewhere in this volume (Lin, 1987).

Animals↗
PubMed1987

Structure-activity relationships for induction of peroxisomal enzyme activities by phthalate monoesters in primary rat hepatocyte cultures.

Rat hepatocytes were cultured for 70 hours with a series of four isomeric octyl and five isomeric hexyl phthalate monoesters, and their effects on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) and carnitine acetyltransferase activities determined. All nine monoesters produced dose-related increases in enzyme activities and marked quantitative compound potency differences were observed. Generally octyl isomers were more potent than hexyl isomers and 2- and 3-ethyl substituted isomers were more potent than their straight chain and 1-ethyl substituted analogs. For example, mono(2-ethylhexyl)phthalate was more potent than mono(1-ethylhexyl)phthalate, and this was also observed after oral administration of the two isomers to rats for seven days. The cell culture data for induction of palmitoyl-CoA oxidation were used to generate quantitative structure-activity relationships. Relatively poor correlations were observed between biological activity and simple hydrophobic parameters, but a good correlation was obtained when compound electronic structural parameters, obtained by molecular orbital calculations, were employed. These studies demonstrate relationships between biological activity and chemical structure for a series of phthalate monoesters and indicate the potential usefulness of primary rat hepatocyte cultures to screen compounds for peroxisome proliferation.

Animals↗
PubMed1986

Structure activity studies on the induction of peroxisomal enzyme activities by a series of phthalate monoesters in primary rat hepatocyte cultures.

A series of 9 phthalate monoesters were cultured with primary rat hepatocytes for 70 h and their effect on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) determined. Marked quantitative differences in the induction of enzyme activity were observed with both alkyl chain length and position of side chain substitution affecting compound potency. With all 9 compounds a good correlation was observed between electronic structural parameters obtained by molecular orbital calculations and biological activity in the cell culture system. These results demonstrate a relationship between chemical structure and biological activity for a series of phthalate monoesters and indicate the potential usefulness of primary hepatocyte cultures to screen compounds for peroxisome proliferation.

Acyl Coenzyme A↗
PubMed1986

An ultrastructural study of ethylene glycol monomethyl ether-induced spermatocyte injury in the rat.

Previous studies have shown that administration of ethylene glycol monomethyl ether (EGM) to the rat results in testicular damage with the spermatocyte being the primary cellular site for toxicity. An ultrastructural study has now been carried out to characterize the early subcellular changes following a single dose of 250 or 500 mg EGM/kg body weight. At 12 and 18 hr after dosing, large membrane bound intracellular vacuoles filled with flocculent or granular material were observed in the basal region of the tubules. These mostly appeared to originate as rarefaction and swelling of Sertoli cell processes. Necrosis of spermatocytes was evident at 12 hr but the process of cell death was rapid with cells either appearing normal or in an advanced state of necrosis. Many of the spermatocytes which appeared otherwise normal, showed areas of plasma membrane dissolution, a change which was also seen affecting the facing Sertoli cell plasma membrane resulting in continuity of the cytoplasm between the two cells. This membrane change was seen in the absence of any other morphological abnormality in the affected spermatocyte, although slight mitochondrial condensation was sometimes also present. By 24 hr after dosing, most of the spermatocytes in the spermatogenic stages which had shown the earlier membrane changes, had become necrotic while Sertoli cell vacuolation had largely regressed. Although the membrane dissolution was an early and specific effect, it is regarded as part of the phagocytic response of the surrounding Sertoli cell. It is suggested that the vacuolar changes in the Sertoli cell may be causally associated with spermatocyte injury.

Animals↗
PubMed1986

Effects of di-(2-ethylhexyl) phthalate and five of its metabolites on rat testis in vivo and in in vitro.

In an attempt to establish which compound or compounds are responsible for the testicular damage observed after administration of di-(2-ethylhexyl) phthalate (DEHP) in rats, the effects of the parent compound and five of its major metabolites (mono-(2-ethylhexyl) phthalate (MEHP), 2-ethylhexanol (2-EH), mono-(5-carboxy-2-ethylpentyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate) were investigated in vivo and in vitro. The concentrations of MEHP and the three MEHP-derived metabolites in plasma were determined after single and multiple oral doses of DEHP. The plasma concentrations and areas under the plasma concentration-time curves (AUC's) of each of the MEHP-derived metabolites were considerably lower than those of MEHP both after single and after repeated administration of 2.7 mmol of DEHP/kg body weight. The mean elimination half-life of MEHP was significantly shorter in animals given repetitive doses than in those given a single dose, but there was no statistically significant difference between the mean AUC values. No testicular damage was observed in young rats given oral doses of 2.7 mmol of DEHP or 2-EH/kg body weight daily for five days. In animals which received corresponding doses of MEHP the number of degenerated spermatocytes and spermatids was increased, whereas no such effects were found in animals given the MEHP-derived metabolites. MEHP was also the only compound that enhanced germ cell detachment from mixed primary cultures of Sertoli and germ cells.

Animals↗
PubMed1986

Aspects of the testicular toxicity of phthalate esters.

Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15-week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. [14C]Mono-n-pentyl phthalate and [14C]MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this. Some features of the testicular lesion could be reproduced in primary cocultures of rat Sertoli and germ cells. Structure activity studies with a range of phthalate monoesters showed good agreement between the induction of germ cell detachment in culture and testicular toxicity in vivo. Three metabolites of MEHP (metabolites V, VI, and IX) were much less toxic in culture than MEHP itself, suggesting that the latter may be the active testicular toxin from DEHP.

Age Factors↗
PubMed1986

Hepatic effects of phthalate esters and related compounds--in vivo and in vitro correlations.

The hepatic effects of phthalate esters and related compounds on peroxisomal and microsomal enzyme activities were investigated in the intact animal and in primary hepatocyte cultures. In vitro studies with a series of phthalate monoesters demonstrated structure activity differences in the induction of the specific peroxisomal marker KCN-insensitive palmitoyl-CoA oxidation and also of carnitine acetyltransferase. These effects could be reproduced in vivo, and potency differences were also observed between di(2-ethylhexyl) phthalate (DEHP) and its straight-chain isomer, di-n-octyl phthalate. In in vivo studies, DEHP, mono(2-ethylhexyl) phthalate, and clofibrate/clofibric acid produced large increases in liver size and peroxisomal enzyme activities in Sprague-Dawley rats and Chinese hamsters, but had less effect in Syrian hamsters. These effects could be largely reproduced in vitro where good responses were obtained with rat and Chinese hamster hepatocytes, but either little or no effect with Syrian hamster and Dunkin-Hartley guinea pig hepatocytes. Both DEHP and clofibrate appeared to induce similar form(s) of microsomal cytochrome P-450 which exhibited a high specificity towards lauric acid hydroxylation. With a range of hypolipidemic agents, including phthalate monoesters, a good correlation was observed between the induction of peroxisomal and microsomal enzyme activities in rat hepatocyte cultures. These results thus demonstrate a good relationship between the in vivo and in vitro effects of phthalate esters and related compounds and suggest that hepatocyte cultures may be useful for further studies on the hepatic effects of peroxisome proliferators.

Animals↗
PubMed1985

Effect of very long chain fatty acids on peroxisomal beta-oxidation in primary rat hepatocyte cultures.

Previous studies have demonstrated that certain high fat diets can induce peroxisomal fatty acid beta-oxidation in rodent liver and that this may be due to their content of trans 22:1 fatty acids. In this study we have examined the effects of cis and trans 22:1 fatty acids (erucic and brassidic) and oleic acid (18:1) on palmitoyl-CoA oxidation, carnitine acetyltransferase and carnitine palmitoyltransferase activities in primary rat hepatocyte cultures. Brassidic and erucic acid and, to a lesser extent, oleic acid were cytotoxic to rat hepatocytes. However, at a concentration of 0.1 mM, brassidic acid produced small increases in palmitoyl-CoA oxidation and carnitine acetyltransferase activities in hepatocytes cultured 70 hr. Treatment of cells with 0.1 and 0.3 mM of either erucic or oleic acid had no effect on any of the enzymes measured.

Animals↗
PubMed1985

A quantitative study of stage-specific spermatocyte damage following administration of ethylene glycol monomethyl ether in the rat.

A quantitative study has been carried out to characterize the stage susceptibility of the spermatocyte to ethylene glycol monomethyl ether (EGM) toxicity. EGM was administered as a single oral dose of 250 mg/kg body wt and rats were examined at time periods after dosing. The number of spermatocytes and round spermatids in tubules at each stage of spermatogenesis was counted. A sharp transition in susceptibility was observed between zygotene spermatocytes in stage XIV which showed no effect and pachytene spermatocytes in stage I which showed death or depletion of 70% of its population after 1 day. A similar transition was seen between dividing spermatocytes and step 1 spermatids, the latter being unaffected. There was a gradual reduction in susceptibility toward midpachytene such that cells in stages VII-XI showed no effect. Analysis of later time periods revealed no effect on spermatogonia or prepachytene spermatocytes but did indicate that midpachytene spermatocytes underwent delayed cell death after further progression through the cycle. In a separate sequential morphological study of early changes, the earliest signs of necrosis were seen 12 hr after dosing and were restricted to spermatocytes in stages V, XI, and XII. Cell death then progressed in a wave-like manner through stages XIII and XIV finally reaching stage I, 24 hr after dosing.

Animals↗
PubMed1985

The role of metabolism in 2-methoxyethanol-induced testicular toxicity.

The role of metabolism in 2-methoxyethanol (ME)-induced testicular toxicity has been investigated with Sprague-Dawley rats. Following administration of [14C]ME (250 mg/kg, ip) to a group of animals, there was evidence of testicular damage, identified as depletion of the spermatocyte population. Radioactivity detected in urine over 48 hr after treatment accounted for 55% of the dose. The major urinary metabolites were identified by HPLC and isotope dilution analysis, as methoxyacetic acid (MAA) and methoxyacetylglycine (accounting for 50 to 60% and 18 to 25%, respectively, of urinary radioactivity). Analysis of plasma revealed a rapid conversion of ME to MAA (t1/2 for disappearance of ME = 0.6 +/- 0.03 hr) and gradual clearance of radioactivity (t1/2 = 19.7 +/- 2.3 hr). Pretreatment of animals with pyrazole (400 mg/kg, ip) 1 hr prior to [14C]ME dosing gave complete protection against the testicular toxicity of ME. Radioactivity detected in the urine from the pyrazole-pretreated groups over 48 hr (18%) was significantly lower than in the ME-only group. The major radioactive peak co-chromatographed with ME (30 to 36% of the total urinary radioactivity). MAA and methoxyacetylglycine were not major metabolites. Analysis of plasma revealed almost complete inhibition of the conversion of ME to MAA (t1/2 for disappearance of ME = 42.6 +/- 5.6 hr, clearance of radioactivity t1/2 = 51.0 +/- 7.8 hr). The results demonstrate that metabolic activation is required for 2-methoxyethanol to exert toxicity to the male reproductive system.

Acetates↗
PubMed1985

Studies on the toxicity of some glycol ethers and alkoxyacetic acids in primary testicular cell cultures.

Primary mixed cultures of Sertoli and germ cells were prepared from testes of immature rats and their response to the known testicular toxicants ethylene glycol monomethyl ether (EGM) and ethylene glycol monoethyl ether (EGE) was studied. Neither EGM nor EGE produced any morphological evidence of toxicity when added to the culture medium at up to 50 mM for 72 hr. In contrast, their metabolites methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) at 2 to 10 mM for 24 to 72 hr caused degeneration of the pachytene and dividing spermatocytes, the target cells of the parent ethers in vivo. As in vivo, earlier spermatocytes, spermatogonia, and Sertoli cells appeared unaffected. EAA was less potent than MAA whereas n-propoxy- and n-butoxyacetic acid, and methoxyacetylglycine, a further metabolite of MAA, produced no morphological changes under these conditions. The same order of toxicity was observed in concurrent studies with the four acids in rats. In culture, the severity of the morphological changes was paralleled by decreases in the activity of carnitine acetyltransferase and lactate dehydrogenase-X in the attached germ cell fraction. Analysis of culture medium provided no evidence for the conversion of EGM to MAA or other metabolites or for the further metabolism of MAA. The close correspondence between the testicular toxicity of alkoxyacetic acids in culture and in vivo suggests a similar mode of action in both cases and points to the potential value of these cultures for mechanistic studies and for screening purposes. The results also emphasize the role of metabolism in the testicular toxicity of glycol ethers and indicate that MAA is an active metabolite of EGM.

Acetates↗
PubMed1985

A simple re-embedding method for the preparation of testicular cell cultures for light and electron microscopy.

A simple and reproducible method is described for the preparation of cell cultures for light and transmission electron microscopy. Mixed testicular cells are grown on collagen layered, PTFE-coated coverslips. Thin sheets of plastic-embedded cells are packed together and re-embedded permitting the examination of sections cut perpendicular to the plane of the culture and, thus, the intercellular relationships at different levels throughout the culture.

Animals↗
PubMed1984

The effect of peroxisome proliferators on the metabolism and spectral interaction of endogenous substrates of cytochrome P-450 in rat hepatic microsomes.

The peroxisome proliferators clofibric acid and di-(2-ethylhexyl)-phthalate (DEHP) preferentially induced the 12-hydroxylation, compared to the 11-hydroxylation, of lauric acid in rat liver microsomes. A marked increase in the affinity of spectral interaction of this substrate with cytochrome P-450 was also observed. In addition, both clofibric acid and DEHP treatment produced a marked effect on the profile of site- and stereo-specific microsomal metabolites of testosterone. These results demonstrate that both peroxisome proliferators induce similar form(s) of cytochrome P-450 which are active in the metabolism of endogenous substrates of cytochrome P-450. The possible relevance of these findings to the hepatotoxicity of peroxisome proliferators is discussed.

Animals↗
PubMed1984

Comparative studies on di-(2-ethylhexyl) phthalate-induced hepatic peroxisome proliferation in the rat and hamster.

Young male Sprague-Dawley rats and Syrian hamsters were treated with 25-1000 mg/kg/day di-(2-ethylhexyl) phthalate (DEHP) orally for 14 days. Liver enlargement was observed in both species, the magnitude being greater in the rat than in the hamster. In the rat there was a marked dose-dependent induction of the peroxisomal marker cyanide-insensitive palmitoyl-CoA oxidation and also of carnitine acetyltransferase. Little effect was observed on the mitochondrial markers carnitine palmitoyltransferase and succinate dehydrogenase. Whereas in the rat, increased peroxisomal enzyme activities were observed after treatment with 100 and 250 mg/kg/day DEHP, much less effect was observed in the hamster even after 1000 mg/kg/day DEHP. Parallel morphological investigations demonstrated a greater increase in hepatic peroxisome numbers in the rat than in the hamster. 14C-labeled DEHP was found to be more rapidly hydrolyzed by rat than hamster hepatic and small intestinal mucosal cell preparations and differences were also observed in the absorption and excretion of oral doses of [14C]DEHP. Studies with mono-(2-ethylhexyl) phthalate (MEHP), a primary metabolite of DEHP, and a hypolipidemic drug clofibrate also resulted in a greater increase in hepatic peroxisomal enzymes in the rat compared to the hamster. The results demonstrate that while DEHP, MEHP, and clofibrate induced hepatic peroxisome proliferation in both species, there was a marked species difference in response. Comparative long-term studies in these species may thus help to clarify the role of peroxisome proliferation in the hepatocarcinogenicity of DEHP.

Animals↗
PubMed1984

Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells.

Mixed cultures of Sertoli and germ cells were prepared from rat testes and their response to some model testicular toxins was studied. Cultures consisted of a monolayer of Sertoli cells to which clusters of spermatocytes and spermatogonia adhered. With time in culture, germ cells progressively detached from the Sertoli cells into the medium. Addition of mono-(2-ethylhexyl) phthalate (MEHP) to the culture medium resulted in a concentration-dependent increase in the rate of germ-cell detachment over the range 10(-7) - 10(-4) M. No such effect was produced by di-(2-ethylhexyl) phthalate or 2-ethylhexanol. An increased rate of germ-cell detachment was also produced by other phthalate monoesters known to cause testicular damage in vivo, whereas similar concentrations of a number of monophthalates not known to affect the testis in vivo had no such effect on the cultures. Known age and species differences in the testicular toxicity of di-(2-ethylhexyl) phthalate could be reproduced in cultures treated with MEHP. There was little effect on the viability of either germ cells or Sertoli cells at concentrations of MEHP that caused marked germ-cell detachment, but there were changes in Sertoli-cell morphology. Increased germ-cell detachment was also observed in cultures treated with 10(-7) - 10(-4) M-AF1312/TS, a compound that affects Sertoli cells in vivo, but was not seen in cultures exposed to a range of other testicular toxins with different target cells in the testis. Thus, the effects produced by phthalate monoesters in vitro may reflect damage to the Sertoli cells. Testicular cell cultures could be of value both for screening compounds and for studying underlying mechanisms of testicular toxicity.

Animals↗
PubMed1984

Comparative studies of the hepatic effects of di- and mono-n-octyl phthalates, di-(2-ethylhexyl) phthalate and clofibrate in the rat.

The oral administration of di-n-octyl phthalate (DNOP), mono-n-octyl phthalate (MNOP), di-(2-ethylhexyl) phthalate (DEHP) and clofibrate to young male Sprague-Dawley rats for 14 days resulted in liver enlargement. Morphological examination of liver sections from DEHP and clofibrate treated rats, but not from either DNOP or MNOP treated animals, revealed increased numbers of peroxisomes (microbodies). Both DEHP and clofibrate treatment markedly stimulated the activities of certain peroxisomal marker enzymes whereas DNOP and MNOP produced only marginal effects. Similarly both DEHP and clofibrate, but not DNOP or MNOP, increased microsomal cytochrome P-450 content and markedly stimulated microsomal lauric acid hydroxylation activity. The results thus demonstrate that whilst the branched chain phthalate ester DEHP induced peroxisomal proliferation, the straight chain analogue DNOP and its metabolite MNOP were essentially inactive. In addition, DEHP treatment appeared to induce similar form(s) of cytochrome P-450 in rat liver to those previously described after clofibrate administration.

Animals↗
PubMed1984

Testicular toxicity produced by ethylene glycol monomethyl and monoethyl ethers in the rat.

Ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE) were administered orally to young male rats at doses varying from 50 to 500 mg/kg/day and 250 to 1000 mg/kg/day for EGME and EGEE, respectively, for 11 days. At sequential times animals were killed and testicular histology examined. The initial and major site of damage following EGME treatment was restricted to the primary spermatocytes undergoing postzygotene meiotic maturation and division. EGEE produced damage of an identical nature, but a larger dose was required to elicit equivalent severity (500 mg EGEE/kg being approximately equivalent to 100 mg EGME/kg). Additionally, within the spermatocyte population, differential sensitivity was observed depending on the precise stage of meiotic maturation: dividing (stage XIV) and early pachytene (stages I-II) greater than late pachytene (stages VIII-XIII) greater than mid-pachytene (stages III-VII). Equivalent doses of methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) gave injury similar to the corresponding glycol ether. When animals were pretreated with inhibitors of alcohol metabolism followed by a testicular toxic dose of EGME (500 mg/kg), an inhibitor of alcohol dehydrogenase (pyrazole) offered complete protection. Pretreatment with the aldehyde dehydrogenase inhibitors disulfiram or pargyline did not ameliorate the testicular toxicity of EGME. In mixed cultures of Sertoli-germ cells, MAA and not EGME produced effects on spermatocytes analogous to that seen in vivo, at concentrations approximately equivalent to steady-state plasma levels after a single oral dose of EGME (500 mg/kg). It would seem likely that a metabolite (MAA or possibly methoxyacetaldehyde) and not EGME is responsible for the production of testicular damage.

Acetates↗