Heteroclitic immunization induces tumor immunity.

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Tumor immunity and autoimmunity induced by immunization with homologous DNA.

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.

Priming for T-cell-mediated rejection of established tumors by cutaneous DNA immunization.

DNA immunization has been shown to elicit both antibody and CTL responses against antigens expressed by infectious organisms. Because CTL responses have been implicated in rejection of cancer, we investigated whether DNA immunization by particle bombardment using a gene gun could induce CTL responses that were capable of rejecting tumors in mice. DNA immunization by particle bombardment using genes encoding beta-galactosidase and ovalbumin primed mice to generate CTLs in two genetic backgrounds (DBA/2 and C57BL/6 strains, respectively). DNA immunization was more potent in inducing CTLs than immunization with an optimized regimen of ovalbumin peptide plus immune adjuvant. Immunity induced by DNA immunization protected mice against s.c. challenge with tumors expressing the beta-galactosidase antigen. Tumors were rejected even when DNA immunization was started 3 or 7 days after tumor challenge as tumors were becoming established. Tumor rejection required CD8(+) T cells, confirming a role for CTLs in vivo. These studies show that DNA immunization by particle bombardment can efficiently induce CTL responses that are capable of rejecting even established tumors.

The penetration of 2,3,7,8-tetrachlorodibenzo-p-dioxin into viable and non-viable porcine skin in vitro.

Freshly harvested, full thickness porcine skin was kept metabolically viable at 4 degrees C in a minimal essential medium for at least 48 h, as judged by the formation of lactate or 14CO2 from 14C-labeled glucose. In vitro topical exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 65 ng/cm2) for up to 1000 min did not affect the viability of skin. The penetration and distribution of TCDD into porcine skin was studied in an in vitro system under a variety of conditions, such as viability status, different vehicles or concentrations, or artificial removal of the stratum corneum. Loss of viability of the skin increased the rate of penetration of TCDD by about 60%. Removal of the stratum corneum to mimic lesioned skin increased the rate of dermal penetration of TCDD about 3-fold. The use of acetone as the vehicle, simulating dermal exposure to TCDD as a dust or from a volatile solvent, resulted in higher rates of penetration than the use of mineral oil as the vehicle, which simulates the situation of industrial accidents. The percentage of dose absorbed was independent of the dose of TCDD (65 or 6.5 ng/cm2) administered to the surface of skin. Rates of dermal penetration of TCDD ranged form 14 to 985 pg/cm2 skin per h, or 0.2-1.5% of the dose/h, depending on the conditions of exposure. These rates of penetration are comparable with results obtained by others in several other species, with both in vitro and in vivo systems including human skin in vitro. Full thickness porcine skin, viable or previously frozen, is therefore a valid in vitro model to estimate dermal penetration of TCDD in humans.

Reduced activity of tryptophan 2,3-dioxygenase in the liver of rats treated with chlorinated dibenzo-p-dioxins (CDDs): dose-responses and structure-activity relationship.

The activity of tryptophan 2,3-dioxygenase (TdO) was measured in the livers of male Sprague-Dawley rats after acutely toxic doses (LD20-LD80) of chlorinated dibenzo-p-dioxins (CDDs) with 4 of the up to 7 chlorine substituents occupying the 2,3,7,8-positions. Treatment with toxic doses of CDDs results in voluntary feed refusal of rats. A corresponding involuntary reduction of feed intake in naive animals (pair-feeding) causes elevated levels of TdO activity. In the CDD treated rats, however, TdO activities were dose-dependently reduced. An LD80 reduced TdO activity to about 50% of the level found in the corresponding pair-fed animals. This decrease of TdO activity explains the dose-dependent increase of serum tryptophan, which in turn is the likely cause of voluntary feed refusal observed in CDD-treated rats. The activity of another enzyme which is regulated in a fashion very similar to that of TdO, viz., tyrosine aminotransferase (TAT), was consistently, but not dose-dependently, affected by treatment with CDDs.

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA levels.

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 micrograms/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate that the decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.

The enzyme inducers 3-methylcholanthrene and phenobarbital affect the activities of glucocorticoid hormone-regulated enzymes in rat liver and kidney.

3-Methylcholanthrene, an inducer of P448-type cytochromes (mostly 1A1 and 1A2), and phenobarbital, an inducer of P450-type cytochromes (mostly 2B1 and 2B2), are prototypical for the actions of many xenobiotics. They cause endocrine disruption by affecting, among others, steroid hormone levels. Rats were treated with single bolus doses of 3-methylcholanthrene or phenobarbital, and enzyme activities that are controlled by glucocorticoids were measured in liver and kidney. The activities of the cytosolic enzymes L-alanine aminotransferase, indoleamine 2,3-dioxygenase (L-tryptophan pyrrolase), phosphoenolpyruvate carboxykinase, L-serine dehydratase and L-tyrosine aminotransferase were affected in a similar fashion: an initial activity reduction followed by two overshoots of activity 1 and 2 days after dosing. 3-Hydroxy-3-methylglutaryl coenzyme A reductase, the microsomal key enzyme of sterol synthesis, responded with a temporary reduction of activity only and evidently lost its diurnal rhythm. The time course of these changes is most likely caused by a combination of sub-physiological levels of glucocorticoids plus changes of other regulatory hormones elicited by feed intake, postprandial state, etc. A possible role for a combined action of the arylhydrocarbon (Ah) and glucocorticoid receptors in the effects of 3-methylcholanthrene is also suggested.

Reduced activities of key enzymes of gluconeogenesis as possible cause of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats.

Male Sprague--Dawley rats (350-375 g) were injected i.p. with TCDD (25 [sublethal dose] and 125 micrograms/kg [lethal dose], respectively, in corn oil/acetone), or vehicle only; vehicle-treated animals were pair-fed to their TCDD-treated counterparts. 1, 2, 4, 8, 16, and 32 days (28 days for lethal dose) thereafter, animals were sacrificed and activities of two key enzymes of gluconeogenesis determined in livers of rats. In livers of pair-fed rats both enzyme activities were little affected. In the livers of TCDD-treated animals the activity of phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) decreased rapidly, exhibiting significant losses by the 2nd day after treatment. Time course and extent of loss of PEPCK activity (about 50%) were similar after either dose. The activity of glucose-6-phosphatase (G-6-Pase, EC 3.1.3.9) decreased more slowly as a result of TCDD treatment; statistically significant losses were observed by 4 or 8 days after the lethal and sublethal dose, respectively. These results confirm the hypothesis that reduced in vivo rates of gluconeogenesis in TCDD-treated rats are due to decreased activities of gluconeogenic enzymes. In an additional set of experiments, rats were treated with 125 micrograms/kg TCDD, 25 micrograms/kg TCDD, or with vehicle alone. The 25 micrograms/kg or vehicle-treated rats were then pair-fed to rats dosed with 125 micrograms/kg of TCDD. Mean time to death and body weight loss at the time of death were essentially identical in all groups, lending additional support to the hypothesis that reduced feed intake is the major cause of TCDD-induced death in male Sprague--Dawley rats. Both appetite suppression and reduced total PEPCK activity in whole livers occurred in the same dose-ranges of TCDD, suggesting the possibility of a cause-effect relationship.

Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

The relationship between thymic atrophy and plasma corticosterone levels was examined in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated, pair-fed and ad libitum-fed male Sprague-Dawley rats given a usually lethal (125 micrograms/kg) or non-lethal (25 micrograms/kg) dose of TCDD. At both dosages, corticosterone levels in TCDD-treated animals begun to rise as early as day 4 after treatment. At later time points corticosterone levels were 5-7 times higher in rats given the non-lethal dose, and 6-10 times higher in rats administered the lethal dose than the levels observed in ad libitum-fed controls. Corticosterone levels in control rats pair-fed to the lethal dose group (as a result of the severe reduction in feed intake) were similarly elevated as in TCDD-treated rats but this was not the case in pair-fed rats of the non-lethal TCDD dosage (due to an essentially unchanged feed intake). At both dosages, relative thymus weights of TCDD-treated rats started decreasing by day 4 and continued to decline for the most part of the study. Relative thymus weights of rats pair-fed to the non-lethal TCDD dosage were not different from ad libitum-fed rats. However, the decrease in relative thymus weights of rats pair-fed to the lethal TCDD dosage paralleled that of TCDD-treated rats with an apparent 8-day lag period. Morphologically, the thymus as well as the adrenal revealed differential changes in TCDD-treated rats from those observable in pair-fed rats. These results suggest that either TCDD exerts a direct effect on the thymus and the adrenals or it causes an additional stress (e.g., a metabolic stress) over and above the starvation stress, which may be responsible for the differential morphological changes in these glands.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thermogenesis in brown adipose tissue of rats.

Male Sprague-Dawley rats were treated with a usually lethal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 125 micrograms/kg i.p. in corn oil) or with vehicle alone. Two, 4, and 8 days after treatment the temperature of interscapular brown adipose tissue (IBAT) was monitored during venous infusion of norepinephrine (480 ng/min) for 60 min. The temperature response was about 1.0-1.5 degrees C within 1 h in vehicle-treated, pair-fed and ad libitum-fed controls. In TCDD-treated animals, the response of IBAT decreased with time after TCDD dosage, amounting to only 0.3 +/- 0.1 degree C at 8 days after dosing (differences significant with respect to both controls, P less than 0.05). GDP binding to IBAT mitochondria (a measure of thermogenic capacity) was unchanged in all groups, indicating that the reduced thermogenic response was probably not caused by an impairment of the mitochondrial uncoupling process by TCDD.

The effect of gamma-hexachlorocyclohexane (lindane) on the activities of liver lipogenic enzymes and on serum lipids in rats.

The effect of dietary gamma-hexachlorocyclohexane (lindane) (50-350 ppm, 0.17-1.19 mumol/kg chow) on the activity of enzymes of lipogenesis, viz., fatty acid synthase (FAS; EC 2.3.1.85), citrate cleavage enzyme (CCE; EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44), and on serum lipid levels, was investigated in livers of 35-day-old male Wistar rats. Lindane (150 ppm) caused a substantial decline of enzyme activities within the first 24 h of treatment. The decrease was transient, however, and enzyme activities subsequently recovered despite continuation of lindane feeding. The recovery of enzyme activities was comparatively fast in the case of ME, G6PDH and PGDH, but very slow with FAS and CCE. Activities of lipogenic enzymes decrease when animals are starved, and increase much beyond prestarvation levels upon subsequent refeeding. Lindane in the refeeding diet blunted this overshoot of FAS and CCE activities in a dose-dependent manner. In contrast, activities of Me, G6PDH and PGDH responded to low dietary lindane concentrations with a substantial stimulation of the increase of activity, whereas at high lindane concentrations the overshoot was inhibited. According to their responses to lindane exposure, liver lipogenic enzymes could be grouped into 2 categories with FAS and CCE representing one and ME, G6PDH and PGDH representing the other group. Polychlorinated biphenyls (PCBs) in the diet caused basically opposite changes of the activities of the lipogenic enzymes. Co-administration of lindane and PCBs resulted in an apparent cancellation of effects, suggesting that lindane and PCBs affect fatty acid synthesis at opposite points.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutritional regulation of the activities of lipogenic enzymes of rat liver and brown adipose tissue.

Nutrition-induced effects on the activity of enzymes of lipogenesis, fatty acid synthase (FAS: EC 2.3.1.85), ATP citrate lyase (ACL: EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44) were investigated in liver and interscapular brown adipose tissue (BAT) of rats. The lipogenic enzymes could be grouped into two categories according to their response to dietary manipulations: FAS and ACL, both key enzymes of lipogenesis, responded fast and strongly to dietary manipulations. ME, G6PDH and PGDH, enzymes which also contribute to metabolic pathways other than lipogenesis, responded in a more sustained and less pronounced fashion. Feed deprivation caused the specific activities of lipogenic enzymes to decline several-fold. Refeeding of previously fasted (up to 3 days) animals increased the activities dramatically (10-to 25-fold) to far above pre-fasting levels ("overshoot"). Repetition of the fasting/refeeding regimen increasingly impaired the ability of both tissues to synthesize overshooting enzyme activities in the subsequent refeeding period. The fasting-induced decline of the activities was prevented when sugars were provided to the animals via drinking water. The sugars displayed different effectivities: sucrose = glucose > fructose > maltose > > lactose. Sugars as the sole nutrient after fasting were also able to induce overshooting enzyme activities. Again, activities of FAS and ACL responded in a more pronounced fashion than the other three enzymes. Transition from feeding one diet to feeding a new diet of different composition led to adaptation of the lipogenic enzyme activities to levels characteristic for the new diet. Replacing a low-carbohydrate with a high-carbohydrate diet proceeded with major alterations of enzyme activities. This process of attaining a new level took up to 20 days and involved pronounced oscillations of the specific activities. In contrast, when a high-carbohydrate diet was replaced with another diet. particular one high in fat, transition to new enzyme activities was completed within 2-3 days and proceeded without oscillations. All dietary manipulations caused more pronounced responses in young (35d-old) than in adult (180d-old) animals.

The response of rat serum lipids to diets of varying composition or contaminated with organochlorine pesticides.

The effects of different diets (high carbohydrate, high protein, high fat) and diets contaminated with polychlorinated biphenyls (PCBs) and/or gamma-hexachlorocyclohexane (lindane) on the levels of serum triglycerides, cholesterol and phospholipids were investigated in Wistar rats. Serum triglyceride levels differed significantly among the diets, while those of cholesterol and phospholipids were much less affected by the diet composition. A change in diet composition resulted in a gradual adaptation to the lipid levels characteristic of the new diet with major variations including oscillations. There was, however, no specific component of a diet that could be associated with any specific change in serum lipids. While feed deprivation decreased the serum lipids (40-65% in 3 days), refeeding the starved animals caused pronounced increases of the lipids that were different among the diets. The response of the triglyceride levels was the strongest (up to 10 times the starvation levels) followed by those of the phospholipids (4-fold) and cholesterol (2.5-fold). Response of the triglyceride levels peaked within 1 or 2 days of refeeding, whereas those of cholesterol and phospholipids took 4 days to reach the maximum. Feeding PCB-contaminated diets increased the serum lipids in a dose-dependent manner (15-250 ppm). Higher PCB concentrations were increasingly inhibitory (350 ppm) or overtly toxic (> 400 ppm). Elevated lipids returned to the starting levels immediately after peaking (triglycerides) or only after several days (cholesterol, phospholipids) but with an earlier onset at lower PCB concentrations. Refeeding starved animals with PCB-contaminated diets also increased the serum lipids dose-dependently. Feeding lindane-containing diets (50-150 ppm) as well as refeeding animals with lindane diets resulted in a considerable increase of the triglyceride levels, while cholesterol and phospholipids increased much less. Higher lindane concentrations (250 ppm) were inhibitory. The outcome on serum lipid levels on feeding diets contaminated with both PCBs and lindane was basically additive.

Lipogenic enzymes of rat liver and adipose tissue. Dietary variations and effect of polychlorinated biphenyls.

The lipogenic enzymes fatty acid synthase (FAS; EC 2.3.1.85), citrate cleavage enzyme (CCE; EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44) were investigated in liver and in brown adipose tissue (BAT) of Wistar rats under various dietary conditions and in the presence of 15 to 250 ppm (approximately 0.045-0.75 mumol/kg chow) polychlorinated biphenyls (PCBs). In response to refeeding starved animals, enzyme activities in both tissues increased to above normal levels and thereafter exhibited pronounced oscillations of their activities. The extent of increase depended on the carbohydrate and fat content of the diet. The lipogenic enzymes could be grouped in two categories according to their sensitivity to dietary carbohydrate: FAS and CCE responded faster to smaller changes in dietary composition, while ME, G6PDH and PGDH required larger changes and more time to respond. Diet-induced alterations of enzyme activities were of the same order of magnitude in liver and BAT. They were age-dependent, being more pronounced in young animals. Independent of the type of dietary manipulations, activities changed in a coordinate fashion, i.e., the changes of the activities of all 5 enzymes occurred at similar ratios to each other with an identical time course. Feeding PCB-containing diets resulted in a considerable increase of the activities of the lipogenic enzymes in liver, which was significantly greater with ME, G6PDH and PGDH. The effect was dose-dependent but transient. In liver the response to PCB feeding was identical in male and female animals, whereas in BAT lipogenic activities increased in females, but decreased in males. Refeeding starved animals with a PCB-containing diet led to an additional stimulation of the normal refeeding-induced increase of the enzyme activities in liver and BAT. This PCB-induced increase was 2-fold for FAS and CCE, but up to 15-fold for the other enzymes. All PCB-induced effects were significantly less pronounced in old than in young animals. In primary hepatocytes activities increased in hormone-free medium in the presence of PCBs. While activity was induced in insuline- and triiodothyronine-containing medium, this increase was significantly greater with PCBs present.

In vivo and in vitro studies on the regulatory link between 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rat liver.

The activities of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase; EC 1.1.1.34), rate-limiting enzyme of cholesterol biosynthesis, and cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), key enzyme of the neutral bile acid synthesis pathway, were measured in the microsomal fraction of rat liver and in rat liver cells to investigate the coordinate regulation of the two pathways. Both enzyme activities exhibited the same diurnal rhythm and responded in a coordinate fashion to fasting or bile acid-feeding (decrease) and to cholestyramine-feeding (increase). Cholesterol-feeding decreased the activity of HMGCoA reductase, increased that of cholesterol 7 alpha-hydroxylase, and concomitantly increased free cholesterol in microsomes. In an ex vivo setting using primary hepatocytes from animals fed a high cholesterol diet the activity of HMGCoA reductase was initially low and that of cholesterol 7 alpha-hydroxylase was elevated. Release of cholesterol into the medium with ongoing incubation caused HMGCoA reductase activity to increase, and that of cholesterol 7 alpha-hydroxylase to decline. Incubation of hepatocytes with a cholesterol-containing lipoprotein fraction stimulated the activity of cholesterol 7 alpha-hydroxylase, but left HMGCoA reductase activity unaffected. The results confirm the idea of a joint regulation of the two key enzymes of cholesterol metabolism in response to the levels of substrate and metabolites, and support the notion that with respect to bile acid and cholesterol levels, respectively, regulation of HMGCoA reductase activity may be secondary to that of cholesterol 7 alpha-hydroxylase. The in vitro studies supply evidence that the effects of cholesterol and bile acid excess or deficiency are direct and do not involve accessory changes of hormone levels or mediators.

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites.

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis.

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.

Hepatocyte damage induced by carbon tetrachloride: inhibited lipoprotein secretion and changed lipoprotein composition.

Changes of lipoprotein secretion and composition in response to CCl4 treatment were studied in monolayer cultures of rat primary hepatocytes. (1) CCl4 decreased secretion of very low density lipoproteins (VLDL) by about 85%, while high density lipoprotein (HDL) secretion was less affected (about 40%). The effect was concentration-dependent. (2) CCl4 significantly inhibited secretion of VLDL- and HDL-associated triglycerides and cholesterol esters. VLDL- and HDL-associated cholesterol was not affected, while secretion of phospholipids was increased. (3) Hepatocytes secreted the apolipoproteins B48, B100, E, C, and A-I. CCl4 reduced secretion of apoproteins associated with VLDL by almost 20%, and by about 75% when associated with HDL. The de novo synthesis of apolipoproteins was attenuated by CCl4. (4) CCl4 caused variations in the apolipoprotein composition in VLDL and HDL. CCl4 intoxication of the liver affected the morphology and/or function of the lipoproteins, which drastically impaired their ability to act as transport vehicles for lipids from the liver to the circulation.

Decontamination of human skin exposed to 2,3,7,8-tetrachlorodibenzo-p-Dioxin (TCDD) in vitro.

Human post-mortem skin was exposed in vitro to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 32 degrees C, under controlled humidity. In one-half of the samples, damage to the surface of the skin was simulated by stripping of the stratum corneum. After incubation with TCDD for 100 min, four different decontamination protocols were performed: (1) the sample was wiped with dry, adsorbent material (cotton balls); (2) a 10-min topical treatment with mineral oil was followed by dry wiping with cotton balls; (3) a 10-min topical treatment with mineral oil was followed by wiping with acetone-soaked cotton balls; and (4) the sample was washed with water and soap. After decontamination, skin samples were incubated (up to 300 min) again at 32 degrees C. One set of both intact and stripped TCDD-exposed skin samples was incubated for 300 min--absent decontamination--and was used as a control. Mineral oil treatment and acetone wipes, or water and soap, were effective in reducing (i.e., about two-fold) the amount of TCDD in the stratum corneum of intact skin. Mineral oil plus dry wipes reduced the amount of TCDD in the stratum corneum by about one-third, whereas dry wiping alone was ineffective. All protocols, however, were similarly effective in reducing the amount of TCDD in the epidermis and upper dermis; TCDD concentrations were decreased locally by factors of up to ten. In the lower dermis, a minimal effect of the decontamination procedures was observed.(ABSTRACT TRUNCATED AT 250 WORDS)