Secretory component as the receptor for polymeric IgA on rat hepatocytes.

Rat hepatocytes in short-term monolayer cultures bound radiolabeled polymeric rat IgA but not IgG. The binding of 125I-IgA was inhibited equally well by unlabeled polymeric IgA and by antiserum to rat secretory component (SC). The antibody to SC, after specific purification and radiolabeling, was bound to hepatocytes as effectively as the IgA. These results indicate that SC acts as the receptor for polymeric IgA on rat hepatocytes as it does on human gut epithelia, and that the transport of IgA from blood to bile in rats across the liver is analogous to that of IgA across human enterocytes.

Sources of the proteins of rat bile.

The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component. The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an alpha2-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.

Endocytic vesicles in liver carry polymeric IgA from serum to bile.

The distributions both of endogenous IgA and of injected 125I-labelled IgA were determined amongst the components of a liver homogenate. Rate zonal sedimentation, under conditions where separation was principally determined by particle size, showed that IgA was tightly bound to material which sedimented in the size range of the larger endoplasmic reticulum fragments. Further fractionation of the components within this size range according to their densities, by isopycnic centrifugation, showed that the IgA was associated with small vesicles with a density range of 1.12--1.17 g/ml, quite distinct from endoplasmic reticulum fragments. We therefore conclude that the IgA is present in liver cells in a distinct class of vesicles, which are, presumably, responsible for the transport of IgA from blood to bile.

Apparent heterogeneity of hepatic lysosomes due to membrane-bound acid phosphatase.

Hepatic lysosomes have been fractionated by rate sedimentation and by isopycnic banding. In all experiments, the distribution of acid phosphatase differed from that of the other lysosomal enzymes. Evidence is presented that this difference is due not to the separation of lysosomes from different cell types, but simply reflects the membrane location of a part of the acid phosphatase.

Effects of mono (2-ethylhexyl) phthalate and its straight chain analogues mono-n-hexylphthalate and mono-n-octyl phthalate on lipid metabolism in isolated hepatocytes.

In cultured hepatocytes, as in vivo, mono-2-ethylhexyl phthalate (MEHP) and its straight chain analogues mono-n-hexyl phthalate (MnHP) and mono-n-octyl phthalate (MnOP) each cause accumulation of lipid but only MEHP produces significant induction of peroxisomal fatty acid oxidizing enzymes. To elucidate the mechanisms underlying this lipid accumulation we investigated the effects of these phthalates and the drug clofibric acid on fatty acid metabolism in suspensions of isolated hepatocytes. The effects were found to be markedly dependent on the nutritional state of the animals from which the hepatocytes were isolated. In hepatocytes isolated from animals fasted overnight, or animals fed ab libitum but killed at approximately 2.30 p.m., MEHP, MnHP, MnOP and clofibric acid each caused a marked rapid stimulation of fatty acid oxidation and the synthesis of triglycerides in hepatocytes when incubated in Hanks saline. Export of very low density lipoprotein (VLDL) from the cells was either unchanged or somewhat reduced. In contrast, in hepatocytes isolated from rats fed ad libitum but killed at approximately 9.30 a.m. MEHP and clofibric acid did not alter fatty acid oxidation or triglyceride synthesis, while MnOP and MnHP increased triglyceride synthesis but decreased fatty acid oxidation. The effects of fasting were largely abolished by incubations of the cells in a complete tissue culture medium (Liebowitz L-15). The results suggest that MEHP and its straight chain analogues can, either as the free acid or the CoA ester, mimic the action of fatty acids in the allosteric regulation of fatty acid metabolism.

Immune responses to chlorpromazine in rats. Detection and relation to hepatotoxicity.

It has frequently been suggested that the jaundice which occurs in a small percentage of human patients following treatment with chlorpromazine is due to a hypersensitivity reaction. It has, however, proved impossible to obtain an animal model for this condition. We now show that oral administration of chlorpromazine at 25 mg/kg per day to Wistar albino rats results in formation of both humoral and secretory antibodies to chlorpromazine. We also demonstrate that the severity of the hepatic changes observed in chlorpromazine-fed animals (periportal glycogen loss and centrilobular fatty change) is enhanced by preimmunization of the rats via the gut-associated lymphoid tissue with a chlorpramizine-protein conjugate. There was, however, no correlation between the titre of either serum or biliary antibodies in individual animals and the degree of liver damage. Our results therefore suggest than an immune mechanism is indeed implicated in chlorpromazine toxicity but show clearly that toxic symptoms are not a simple consequence of the formation of anti-chlorpromazine antibodies.

Effects of phthalic acid esters on the liver and thyroid.

The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P-450 isoenzyme(s) catalyzing omega oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fail to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid. The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.

Changes in rat serum lipoproteins following ligation of the bile duct.

Lipoproteins in the serum of normal and of cholestatic rats have been studied by crossed immunoelectrophoresis coupled with separations by rate and isopycnic density gradient centrifugation and by gel exclusion chromatography. Normal rat serum contained distinct lipoprotein species closely analogous to human VLDL, LDL, HDL2 and HDL3. Three days after ligation of the common bile duct, there were major changes in the lipoproteins of rat serum. The amounts of VLDL, LDL and of a minor HDL component were elevated and several novel types of lipoprotein were detected. Three of these could be identified as the characteristic lipoprotein of cholestasis, LP-X, as an enlarged and modified HDL and as a lipoprotein of density 1.055 g/ml intermediate in size between LDL and VLDL. Some unusually small VLDL particles were also detected. It is concluded that the changes in rat serum lipoproteins following ligation of the common bile duct are very similar to the changes observed in cholestatic disease in human patients.

Bile proteins in the serum of jaundiced rats.

The major bile proteins can be detected by two-dimensional electrophoresis in the serum of cholestatic rats, three days after ligation of the common bile duct. The novel electrophoresis system used agarose gel electrophoresis in the first dimension followed by equilibrium electrophoresis in a polyacrylamide gel gradient.

Reflux of billiary components into blood in experimental intrahepatic cholestasis induced in rats by treatment with alpha-naphthylisothiocyanate.

The sera of rats treated with alpha-naphthylisothiocyanate, an agent reported to cause intrahepatic cholestasis, and of rats treated with carbon tetrachloride, are examined for evidence of leakage of bile into blood. The levels of the four bile components assayed, namely IgA, free IgA secretory component, the biliary isoenzyme of 5'-nucleotidase and of direct reacting (conjugated) bilirubin, were all elevated in the sera of rats treated with alpha-naphthylisothiocyanate. No change in any of these components was found in rats treated with carbon tetrachloride, indicating that their appearance in serum is not an inevitable consequence of damage to hepatocytes. As the bile components assayed reach bile by three different routes. their rapid appearance in the sera of rats treated with alpha-naphthylisothiocyanate indicates reflux of bile into blood, presumably as a result of mechanical obstruction of intraheptic bile ducts.

Bile 5'-nucleotidase in the serum of jaundiced rats.

Rat serum 5'-nucleotidase, L-leucyl-beta-naphthylamidase and beta-glycerophosphatase activities are increased whilst alkaline p-nitrophenylphosphatase and alkaline phosphodiesterase activities are unchanged or decreased three days after bile duct ligation. Affinity chromatography on an immobilised antiserum raised against highly purified liver plasma membranes showed that although 5'-nucleotidase in normal serum is unrelated to the 5'-nucleotidase of liver plasma membrane, the 5'-nucleotidase of bile and much of the 5'-nucleotidase in the jaundiced serum are closely related to the plasma membrane enzyme. Since bile is rich in 5'-nucleotidase, the changes in level of this enzyme after bile duct ligation are most simply explained by leakage of bile into the blood; changes in the patterns of the other enzymes are shown to be consistent with this explanation. The jaundiced serum was examined by gel exclusion chromatography and flotation in sucrose gradients for the presence of small fragments of plasma membrane as reported in human jaundiced sera, but no such fragments could be detected three days after bile-duct ligation.

Time and dose study on the response of rats to the hypolipidaemic drug fenofibrate.

Groups of male Wistar albino rats were administered diets containing sufficient fenofibrate to ensure intakes of either 200, 60 or 13 mg/kg/day or sufficient clofibrate to ensure an intake of 400 mg/kg/day. Four rats from each experimental group and 6 control rats were killed, 3, 7, 14 and 28 days, 8, 12 and 20 weeks and 6, 9, 12 and 18 months after commencement of treatment. At all time points livers were subjected to histological, electron microscopic and biochemical examination, the other major abdominal organs were removed for histological examination. A more extensive necropsy was carried out on rats killed after 12 and 18 months. The major alterations were observed in the liver, although there were also morphological changes in the thyroid, pancreas and kidney after prolonged treatment. The hepatic changes followed a distinct time course. Within 24 h of offering diets containing the compounds to the rats there was accumulation of small droplets of lipid, induction of peroxisomal enzymes and of the specific cytochrome P-450 catalysing omega-hydroxylation of fatty acids and an increase in the number of mitotic figures. More slowly developing changes were loss from the centrilobular zone of fat, glycogen and of glucose 6-phosphatase activity. Here maximal changes were observed after 14 days of treatment. A still more slowly developing change was accumulation of enlarged lipid-loaded lysosomes, which was maximal at 26 weeks, accompanied by the development of lipofuscin bodies. Finally, in animals treated for 12 months or more there was evidence for increasing cell turnover as indicated by an increased number of mitotic figures, more dark cells and induction of serum alanine transaminase. The last 2 groups of changes were not observed in rats treated with 13 mg/kg/day of fenofibrate. In general the degree of change in rats treated with 400 mg/kg/day of clofibrate was similar to those found in rats treated with 60 mg/kg/day of fenofibrate.

Lipid accumulation in the livers of chlorpromazine-treated rats does not induce peroxisome proliferation.

Treatment of rats with 25 mg/kg/day of the neuroleptic drug chlorpromazine for periods of 7, 28 or 90 days causes a slow accumulation of lipid in large droplets in centrilobular hepatocytes. There is little or no damage to hepatocytes as assessed by changes in glucose-6-phosphatase activity and by electron microscopy. Furthermore there is no indication of a change in peroxisomal beta-oxidation of fatty acids or in microsomal omega-oxidation of fatty acids. It is, therefore, clear that lipid accumulation in the liver does not automatically induce peroxisomal and microsomal fatty acid oxidising enzymes.

Alterations in the thyroids of rats treated for long periods with di-(2-ethylhexyl) phthalate or with hypolipidaemic agents.

Treatment of rats for periods of 3 months or longer with the hypolipidaemic drugs clofibrate and fenofibrate or with the plasticiser di-(2-ethylhexyl) phthalate causes alterations in the thyroid. The colloid is shrunken and contains calcium-rich inclusions. Electron microscopy shows increases in the number and size of lysosomes, hypertrophy of the Golgi apparatus and dilation of the rough endoplasmic reticulum. These changes are consistent with persistent hyperactivity in the gland.

Effects on male rats of di-(2-ethylhexyl) phthalate and di-n-hexylphthalate administered alone or in combination.

The effects of phthalate esters of branched chain alcohols, typified by di-(2-ethylhexyl)phthalate (DEHP) differ from those of esters of straight chain alcohols typified by di-n-hexyl phthalate (DnHP). The former induce liver enlargement and proliferation of hepatic peroxisomes, while the latter cause no peroxisome proliferation but cause fat accumulation in the liver. Both classes of phthalate esters are hypolipidaemic and cause thyroid changes associated with an increased rate of thyroglobulin turnover. As phthalate esters are used as mixtures, we have examined the effect of mixtures of the compounds. Groups of five male Wistar albino rats were administered either control diet or diets containing either 10000 ppm of DEHP, 10000 ppm of DnHP or 10000 ppm DEHP plus 10000 ppm DnHP for 14 days. Rats receiving diets containing DEHP showed the expected increase in relative liver weight, in "peroxisomal" fatty acid oxidation and in CYP4A1. Serum triglyceride and serum cholesterol were also reduced, and the thyroid showed the histological changes mentioned above. Rats consuming diets containing DnHP showed no increase in relative liver weight and no induction of peroxisomal fatty acid oxidation or CYP4A1. However, there was a marked accumulation of fat in the liver. The fall in serum cholesterol was similar to that in rats treated with DEHP, but the fall of serum triglyceride was more pronounced. Thyroidal changes were again observed. In general, changes in rats treated with a mixture of DEHP and DnHP were very similar to those found with rats treated with DEHP alone. The liver was enlarged, and peroxisomal fatty acid oxidation and CYP4A1 were both induced. The amount of fat in the liver was much less than in rats receiving DnHP alone. Thyroid changes were similar to those in rats receiving the individual compounds. The effect on serum cholesterol seemed additive, but the levels of serum triglyceride were intermediate between the groups receiving the single compounds.

Proteins of guinea-pig bile: selective resorption in the gall bladder.

We have examined and compared the proteins present in guinea-pig bile as collected either from the common hepatic duct or from the gall bladder. Guinea-pig bile, collected from the common bile duct, has a rather low concentration of protein. Detailed examination shows that the concentrations of actively transported proteins such as immunoglobulin A and haptoglobin:haemoglobin complexes are markedly lower than in rats although the concentrations of proteins which, like albumin, leak non-specifically into bile are similar in the two species. We also find that the protein composition of guinea-pig bile is extensively and selectively modified by resorption of protein in the gall bladder.

Enterohepatic circulation of IgA does not occur in rats.

The concentration of IgA in rat bile falls with time after cannulation. This effect is particularly marked in germ-free animals, in which the initial level of IgA is markedly subnormal anyway. The fall suggests that the cannula is draining to the exterior IgA, which would normally be recirculated. However, when germ-free or normal rats were supplied intraduodenally with bile whose IgA carried 125I, no transfer of this labelled IgA from the gut lumen back to the bile could be detected.

Evidence for and possible mechanisms of non-genotoxic carcinogenesis in rodent liver.

Doses of chemicals which induce hepatocellular necrosis usually induce hepatic tumours if the dosing is frequent and is maintained for long periods. Such necrosis is usually evident within 48 h of the first administration. Similarly, chemicals that lead to marked proliferation of peroxisomes in the liver also usually induce hepatic tumours on pretracked regular dosing. For both of these phenomena failure to produce a certain level of effect, or to maintain it for sufficiently long periods, can result in the observation of a non-carcinogenic response. The exact dose/time requirements for carcinogenicity have not been defined and may be species/strain/sex-specific. Some chemicals induce liver enlargement and mitogenesis in the absence of overt hepatotoxic effects. The early phases of hepatomegaly are associated with mitogenic effects that can be measured as cells in S-phase within the first few days of administration. The later stages of hepatomegaly appear to be associated more with cellular hypertrophy. Both effects appear to be threshold-related. Further, sustained hepatomegaly is associated with proliferation of SER and the induction of a range of liver enzymes. These changes (mitogenesis, hepatomegaly, enzyme induction), in isolation, are less definitive indicators of carcinogenicity, but they occur for a sufficient number of liver-specific carcinogens that their role as early indicators is worthy of confirmed study. The major area of study required for all possible early markers of hepatocarcinogenicity is to establish the dose and time dependence of these changes in relation to the eventual appearance of tumours. Finally, the specificity of all these markers require evaluation by the study of appropriate non-carcinogens.

Hepatic nuclear and cytoplasmic effects following intermittent feeding of rats with di(2-ethylhexyl) phthalate.

The effects on rats of intermittent feeding with the peroxisome proliferator and hepatocarcinogen di(2-ethylhexyl) phthalate (DEHP) have been examined. Male Wistar rats were fed for alternate 7-day periods diets containing 20,000 ppm DEHP or the control diet. The rats were examined 3 days after the start or recommencement of administration of the DEHP-containing diet or after 7 days on the control diet. After the commencement or recommencement of feeding with DEHP the expected increases in liver weight and in the number of peroxisomes were found. The increase in liver: body-weight ratio in response to administration of DEHP-containing diets was greater in rats that had been previously exposed to the compound, but re-administration of DEHP had a less marked effect on the increase in peroxisome number. Morphometric analysis showed that administration of DEHP-containing diets resulted in an increase in cell number in the liver and that a fall in the cell number occurred after the rats had been returned to the control diet for 7 days. Analysis of nuclear size gave results consistent with an increase in tetraploid hepatocytes after treatment with DEHP which was reversed when the rats were returned to control diet.

Hepatic and associated response of rats to pregnancy, lactation and simultaneous treatment with butylated hydroxytoluene.

This paper describes changes in the livers of rats fed diets containing butylated hydroxytoluene (BHT) over two generations in two separate studies. BHT did not produce tumours when tested for carcinogenicity in several studies by the conventional way. However, when BHT was given to rats in a two-generation carcinogenicity study, a high incidence of hepatic tumours was found in males but not in female rats of the F1 generation. A sequential study has been carried out to gain an insight into this unexpected finding, paying particular attention to the perinatal period. In the dose-ranging study designed to assess the tolerance of rats to BHT, groups of male and female rats (F0 generation) were fed diets calculated to deliver 0, 500, 750 and 1000 mg/kg body weight/day. Following a loading period of 5 wk the rats were mated. The BHT content of the diet was not adjusted during pregnancy and lactation. Owing to the normal increase in food consumption during lactation, intakes peaked at double the nominal value by 21 days after the birth of pups. At this time the pups (F1) were weaned onto control diet and maintained on it for 4 wk. At birth, the body weights of pups from the BHT-treated dams were comparable to those of the controls but at weaning the body weights of the pups from all three dose levels were less than those of the controls. At the termination of the experiment (4 wk after weaning), the pups from BHT-treated dams still weighed less than those from untreated controls. In the main experiment the F0 generation were fed 0, 25, 100 and 500 mg/kg body weight/day. Their offspring (F1 generation) were weaned on diets containing the same amount of BHT as the respective parents, apart from the group given the highest dose level (500 mg/kg body weight/day). This dose level was reduced to 250 mg/kg body weight/day at weaning in order to conform with previously published findings. The pups from the dams given the highest dose level were maintained on a dietary concentration of 250 mg/kg body weight/day for the entire study. A group of age-matched non-pregnant females was also studied and the results obtained compared with those from pregnant dams. Pups from all groups were examined at day 20 of gestation, at weaning (21 days after birth), and at 4 and 22 wk post-weaning. There were no effects on fertility and no increase in foetal abnormalities at any dose of BHT. Dams receiving BHT at a nominal dose of 500 mg/kg body weight/day showed liver enlargement accompained by induction of pentoxyresorufin O-depentylase and glutathione S-transferase, and proliferation of the endoplasmic reticulum. Pups from these dams were of the same weight at birth as controls but lost weight during the lactation period. This deficit was not recovered by the time the experiment was terminated. Hence, in two independent studies, the only significant finding in rats treated with BHT in utero and during lactation was that the weight gain of pups during lactation was less than expected when dams received at least 500 mg BHT/kg body weight/day. The body weight of pups did not return to normal following a return to a control diet for 4 wk. It is postulated that the retardation in weight gain of the pups could be due to inadequate milk production.