Immunotoxic effects of exposure of rats to xenobiotics via maternal lactation. Part I 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure of lactating female Leeds rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a reduction in the body, spleen and liver weights of their male offspring at 130 days of age. None of the total administered doses (0.2, 1.0 or 5.0 micrograms/kg b.wt over 18 days) induced thymic atrophy in the offspring of either sex as adults. Most of the growth inhibition occurred during the suckling period and the effect was near maximal following maternal exposure to the lowest dose of TCDD. After this dose, at post-natal day 130 the body weights of the female offspring remained depressed, while those of the males had recovered to untreated control values. Maternal exposure to TCDD affected the immunocompetence of the adult offspring: in vitro T cell dependent and T cell independent responses and mitogen induced in vitro production of interleukin 1 (IL-1) and interleukin 2 (IL-2) were suppressed at post-natal day 130. The total dose of TCDD that has to be administered to dams over an 18-day nursing period in order to reduce the humoral responses of their offspring as adults by 50% of the maximum was estimated to be in the range 0.3-1.0 micrograms/kg b.wt to the antigens SRBC, DNP-Ficoll or TNP-LPS and 3.5-3.9 micrograms/kg b.wt. to the antigen LPS.

Immunotoxic effects of prolonged dietary exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of low level exposure of rats to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) on their immune system was investigated Dietary administration to young adult male Leeds strain rats of a total dose of 3 micrograms/kg body weight of TCDD resulted in an exposure duration-dependent reduction of in vitro lipopolysaccharide-induced production of interleukin (IL)-1 in cultures of their splenic macrophages. A 30-day exposure produced approximately 30% suppression and 180-day exposure produced approximately 52% suppression. This reduction did not negatively influence lipopolysaccharide- induced proliferation of B cells, instead an enhancement of B cell proliferation was observed after 30 days exposure. A 180 day exposure significantly suppressed the generation of IL-2 by either concanavalin A or phorbol myristate acetate/calcium ionophore stimulation, and reduced the lectin-induced proliferation of splenic T cells. The 30-day TCDD exposure showed no such immunotoxicity. TCDD at both exposure durations suppressed the expression of the alpha chain of the IL-2 receptor in concanavalin A-activated T cells, without affecting the CD4+/CD8+ ratio. The results suggest that exposure to a low dietary dose of TCDD suppresses the functions of several T cell subsets, some of the immunotoxic effects being produced early, while others require a longer exposure also down-regulates the IL-1 production function of macrophages. A common mechanism of TCDD immunotoxicity may be on the multifunctional signal transduction pathways downstream to the activation of protein kinase C and Ca2+ flux.

Inhibitory effect of a cholecystokinin antagonist on pancreatic carcinogenesis after pancreatobiliary diversion.

The role of cholecystokinin (CCK) has been explored in pancreatic carcinogenesis following pancreatobiliary diversion (PBD), using the specific CCK receptor antagonist CR-1409. Male Wistar rats (n = 80) weighing 70-100 g were given weekly i.p. injections of azaserine (30 mg kg-1 week-1) for 3 consecutive weeks. One week later animals were randomised to receive either PBD or sham PBD and thereafter to receive s.c. injections of either saline or CR-1409 (10 mg kg-1 day-1, 5 days a week). Six months after operation surviving rats were killed as follows: sham + saline 20, PBD + saline 19, sham + CR-1409 14, PBD + CR-1409 11. Cardiac blood was taken for CCK assay and the pancreas was excised for wet weight measurement and quantitative estimation of atypical acinar cell foci (AACF), the precursor of carcinoma. PBD reduced median body weight (3-20% less than shams) but trebled the absolute and relative pancreatic weights (P < 0.001). CR-1409 blunted this adaptive response to PBD, reducing absolute pancreatic weight by 35% (P < 0.005). PBD quadrupled circulating CCK concentrations, regardless of the antagonist treatment. Acidophilic AACF occurred only in rats with PBD. CR-1409 markedly reduced the number of observed acidophilic AACF by 90% (P < 0.001) and the number of foci per pancreas by 93% (P < 0.001). Moreover, CR-1409 reduced the mean focal diameter of each lesion by 18% (P < 0.005), the mean focal volume by 58% (P < 0.05) and the percentage of pancreas occupied by acidophilic foci by 95% (P < 0.001). PBD enhances pancreatic carcinogenesis by causing hypercholecystokininaemia, and CR-1409 largely inhibits this enhancement.

Duodenogastric reflux enhances growth and carcinogenesis in the rat pancreas.

Surgery for peptic ulcer disease may increase the risk of pancreatic cancer. The effect of duodenogastric reflux on pancreatic carcinogenesis was tested, and changes in the circulating levels of cholecystokinin (CCK) and gastrin were measured. Male Wistar rats (n = 40) weighing 250-300 g were randomized to undergo gastrotomy (control) or split gastrojejunostomy (to produce complete duodenogastric reflux) and then to receive azaserine (30 mg/kg/week intraperitoneally) or saline injections for 3 weeks. At 6 months, blood CCK was assayed and the pancreas was excised for quantitative estimation of atypical acinar cell foci (AACF), the precursor lesions of carcinoma. Rats that had undergone split gastrojejunostomy weighed 15-19 per cent less than controls (P < 0.05), but their relative pancreatic weight (mg pancreas per 100 g body-weight) was 52-60 per cent greater (P < 0.001). Acidophilic AACF occurred only in azaserine-treated rats with duodenogastric reflux. Although plasma CCK concentrations were unchanged, split gastrojejunostomy increased basal and postprandial gastrin levels by 98-175 per cent (P < 0.05). Duodenogastric reflux produces sustained hypergastrinaemia and promotes experimental pancreatic carcinogenesis.

Bile acids do not modify the effects of pepsin on the fine structure of human oesophageal epithelium.

Oesophageal mucosal specimens (n = 250) were taken from 25 normal subjects (14 females, 11 males; median age 52 years; range 19-63 years) and incubated in physiological saline, pepsin and bile acid solutions to determine whether conjugated bile acids modify the epithelial cytotoxic action of pepsin. Short (5 min) and long (22 min) incubations were carried out and the results were assessed by transmission electron microscopy. Six different parameters of epithelial damage were scored (0-4) by a single 'blinded' pathologist for each of four epithelial layers. The scores after incubation in saline (pH 7 and titrated to pH 2 with HCl) were not different from those of the controls (P = 0.35). Both pepsin and bile acids (pH 2) caused more damage than saline at pH 2 (P less than 0.001) which was similar for the two substances (P = 0.136). Conjugated bile acids in combination with pepsin (pH 2) did not alter the overall extent or pattern of damage caused by pepsin alone (pH 2); P = 0.142). Conjugated bile acids, in concentrations commonly encountered during gastro-oesophageal reflux, did not appear to modify the cytopathic effects of pepsin on oesophageal mucosal cells in vitro. Conjugated bile acids may not be important in the pathogenesis of oesophagitis in patients with acid/peptic gastro-oesophageal reflux.

Enhancing effect of partial gastrectomy on pancreatic carcinogenesis.

The controversial issue of enhanced pancreatic carcinogenesis following partial gastrectomy has been explored in male Wistar rats (n = 40) weighing 250-300 g. Animals were randomised to receive either 60% distal gastrectomy with Roux-en-Y reconstruction or gastrotomy and resuture (control). Immediately after operation each group was further divided into two subgroups, receiving i.p. injections of either saline or azaserine (30 mg kg-1 wk-1 for 3 weeks). At 15 months blood was obtained at 0, 5, 15 and 30 min after a fatty meal for cholecystokinin (CCK) assay; rats were then killed. Pancreatic wet weight was measured, and histological sections were examined for atypical acinar cell foci (AACF), the putative precursor lesion of carcinoma. There were no significant differences in body weight or pancreatic weight between controls and rats with gastrectomy. Only azaserine-treated rats had acidophilic AACF. Partial gastrectomy substantially increased the number of acidophilic AACF per pancreas (median 26.05 vs 2.09; P less than 0.005), with a 9-fold increase in their volume (P less than 0.005). Basal and postprandial plasma CCK concentrations were higher after gastrectomy than in controls (P less than 0.05). Partial gastrectomy has an enhancing effect on azaserine-induced pancreatic carcinogenesis, probably by means of increased CCK release.

Effects of dietary fatty acids on the early stages of neoplastic induction in the rat pancreas. Changes in fatty acid composition and development of atypical acinar cell nodules.

Diets enriched with fat, especially unsaturated fat, promote experimental pancreatic carcinogenesis, but little is known of the effects of individual fatty acids. The effect of stearic and oleic acid on pancreatic fatty acids and atypical acinar cell nodules (preneoplastic lesions) was studied in 14-day-old weanling male Leeds strain rats (n = 60) given the carcinogen azaserine. Rats were allocated to one of six groups: untreated controls (n = 10), 20% stearic acid diet (n = 10), 20% oleic acid diet (n = 10), carcinogen alone (n = 10), carcinogen plus 20% stearic acid diet (n = 10) or carcinogen plus 20% oleic acid diet (n = 10). Azaserine was administered by intraperitoneal injection in a dose of 30 mg/kg at 2, 3 and 4 weeks of age. When total lipid extracts of pancreas were examined, there was an increase in stearic acid in the stearic acid fed group and an increase in oleic acid in the oleic acid fed group, irrespective of carcinogen treatment. The relative content of all other pancreatic fatty acids was suppressed by feeding oleic acid. At 26 weeks, the number and volumetric indices of pancreatic atypical acinar cell nodules was increased only in rats given azaserine and oleic acid. The enhancing effect of oleic acid on pancreatic carcinogenesis may be associated with pancreatic fatty acid changes.

Pancreatobiliary diversion enhances experimental pancreatic carcinogenesis.

Since compensatory hyperplasia promotes experimental carcinogenesis in the gut, we tested the ability of two surgical models of pancreatic growth to promote pancreatic carcinogenesis. Male Wistar rats (n = 60) weighing 250-300 g underwent pancreatobiliary diversion (PBD), 90% small bowel resection (PSBR) or triple transection and reanastomosis of the small intestine (controls). Postoperatively, each group received azaserine (20 mg kg-1 wk-1 i.p.) for 6 weeks. Surviving rats were killed at 6 months, pancreatic wet weight was measured and histological sections were examined for atypical acinar cell foci (AACF), the putative precursor of carcinoma. Median relative pancreatic weight (mg pancreas/g body weight) was 2.20 for controls (n = 18), 4.08 for PSBR (n = 11) (P less than 0.001) and 6.86 for PBD (n = 16) (P less than 0.001). PSBR did not affect the development of acidophilic AACF, but PBD produced an enormous increase in their number per cm3 (median 96 vs. 0; P less than 0.001) and a 7-fold increase in their volume (P less than 0.001). Both operations cause pancreatic growth, but only PBD promotes carcinogenesis, possibly because of its unique hormonal effect.

Quantitative electron microscopy of carcinogen-induced alterations in hepatocyte rough endoplasmic reticulum. II. Modulation of the effects of 3'MeDAB by adrenalectomy and adrenal corticosteroids.

A quantitative electron microscope study was made of the effects of bilateral adrenalectomy (ADX) and deoxycorticosterone acetate (DOCA) on the state of aggregation of hepatocyte rough ER cisternae in both untreated and 3'MeDAB-fed Leeds strain rats. Disaggregation of hepatocellular rough ER appears to be a common response of the rat liver to hepatocarcinogenic insult, while ADX and DOCA treatment are known to inhibit the chemical induction of neoplasia in this species. In untreated animals both ADX and DOCA significantly increased the mean number of cisternae per array or stack, while an even more pronounced effect was obtained from a combination of the two. The carcinogen 3'MeDAB induced a highly significant loss of aggregation, which was prevented by the combination of ADX and DOCA. In a separate experiment, a single dose of cortisone acetate was also found to partially reverse 3'MeDAB-induced rough ER disaggregation. In the rat hepatocyte, rough ER stacking or aggregation appears to be at least partially under hormonal control.

Effect of fasting on aggregation of hepatocyte rough endoplasmic reticulum in adrenalectomized and 3'MeDAB-treated rats: quantitative electron microscope study.

A method for the quantitative analysis of the degree to which hepatocyte rough endoplasmic reticulum (ER) is arranged into parallel arrays was used to study the effect of fasting on rough ER aggregation in rat liver cells following either bilateral adrenalectomy or administration of the carcinogenic azo dye 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB). One group of male inbred Leeds strain rats was subjected to bilateral adrenalectomy (ADX): after 1 week half of the animals were fasted for 24 h whereupon the whole group was killed. A second group of rats, fed for 4 weeks on a diet containing 0.06% of the carcinogenic azo dye 3'MeDAB, was similarly divided into two groups that were killed either with or without a prior 24-h fast. Untreated control groups of rats, both fasted and unfasted, were also killed. A quantitative electron microscope study was carried out to investigate the effect of each treatment on the degree to which the hepatocyte rough ER was aggregated into parallel arrays. ADX alone was without effect but caused a dramatic fall in rough ER aggregation when combined with fasting. At least as great an effect was induced by 3'MeDAB, with or without fasting, while fasting alone had a significant but much more modest effect than either ADX or the carcinogen. Thus, two disparate treatments induced morphologically identical responses in hepatocyte rough ER. The implications of this are discussed in terms of known interrelations between glucocorticoids and chemical carcinogenesis in the rat liver.

Quantitative electron microscopy of carcinogen-induced alterations in hepatocyte rough endoplasmic reticulum. I. Chronic effect of 3'MeDAB and short-term effects of azo dyes of different carcinogenic potentials.

A new method has been developed for the quantitative analysis of an ultrastructural change in hepatocyte rough endoplasmic reticulum (ER) that is induced by liver carcinogens. Hitherto only subjective observations of this alteration had been made. Male inbred Leeds rats were fed a diet containing 0.06% of the carcinogenic azo dye 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB). Groups of treated rats, together with untreated controls, were sacrificed after 10 days, 4 weeks, 10 weeks and 17 weeks. Samples of liver tissue from each animal and from 10 3'MeDAB-induced hepatocellular carcinomas (HCC), were examined by electron microscopy. A quantitative study was carried out to investigate the effect of chronic exposure to 3'MeDAB upon the state of aggregation of the hepatocyte rough ER into parallel arrays. As early as 10 days after the start of treatment, the rough ER showed a highly significant degree of disaggregation: the mean number of ER cisternae per array fell from the control value of 6.20 to 3.73. This change was sustained throughout the experiment. At 17 weeks, comparison of the mean array size in the HCC cells with that in the surrounding hepatocytes revealed a further significant decline, from 3.46 to 2.12. Further groups of rats were fed other azo dyes for 4 weeks and subjected to the same assay. The carcinogens 4'-methyl-4-dimethylaminoazobenzene (4'MeDAB) and N,N-dimethyl-4-amino-N-acetyl-N-monomethyl-4-aminoazobenzene (DAAMAB) resembled 3'MeDAB with respect to the degree of rough ER disaggregation they induced. In contrast, the non-carcinogen 3'-trifluoromethyl-4-dimethylaminoazobenzene (3'TFMeDAB) had no such effect, while the weak initiator 2-methyl-4-dimethylaminoazobenzene (2MeDAB) induced disaggregation to a lesser degree than the strong carcinogens. At least with the azo dyes used in this study, the extent of rough ER disaggregation appears to be related to hepatocarcinogenesis.

Fine structural alterations induced in the rat hepatocyte by adrenalectomy: influence of deoxycorticosterone acetate or fasting.

Electron microscopy was used to investigate the changes induced by bilateral adrenalectomy (ADX) in rat hepatocytes. Animals were killed at 1 week post-ADX, either with or without a prior 24 h fast. Further studies were carried out at 4 to 5 weeks after ADX to investigate the effect of administering deoxycorticosterone acetate (DOCA) for 4 weeks on the ADX-induced alterations. At 1 week, fasting had a profound effect on the response of hepatocytes to ADX in that either fasting or ADX alone caused proliferation of smooth endoplasmic reticulum (ER) and reduced glycogen but did not alter the rough ER, while ADX together with fasting resulted in the total depletion of glycogen, the virtual absence of smooth ER and the disaggregation of the rough ER into single cisternae. In contrast to the changes seen at 1 week after ADX in unfasted animals, at 4 weeks the hepatocytes contained increased glycogen in large, dense areas and had a scanty smooth ER, suggesting that some endogenous source of steroid hormones active in carbohydrate metabolism becomes available in male ADX rats by this time. This interpretation was consistent with the finding that DOCA-treatment of ADX rats prevented the glycogen accumulation seen at 4 weeks and also induced some smooth ER proliferation.

Induction by paracetamol of bladder and liver tumours in the rat. Effects on hepatocyte fine structure.

Groups of male and female inbred Leeds strain rats were fed diets containing either 0.5% or 1.0% paracetamol by weight for up to 18 months. At the 1.0% dosage level, 20% of rats of both sexes developed neoplastic nodules of the liver, a statistically significant incidence. These rats also showed gross enlargement of their livers and an increase in foci of cellular alteration, the latter also being observed in the low dosage male rats. Papillomas of the transitional epithelium of the bladder developed in all paracetamol-treated groups, and three rats bore bladder carcinomas. However, significant yields of bladder tumours were only obtained from low dosage females and high dosage males. Additionally, 20 to 25% of paracetamol-treated rats developed hyperplasia of the bladder epithelium, which was not coincident with the presence of bladder calculi. A low yield of tumours at various other sites also arose following paracetamol feeding. An electron microscope study of the livers of paracetamol-treated rats revealed ultrastructural changes in the hepatocytes that resemble those that result from exposure to a variety of known hepatocarcinogens.

Histogenesis of pancreatic carcinogenesis in the hamster: ultrastructural evidence.

Pancreatic carcinogenesis in the Syrian hamster, induced by beta-oxidized derivatives of N-nitroso-di-n-propylamine, constitutes a valuable model of human cancer of the exocrine pancreas. In both species the majority of tumors are adenocarcinomas: superficially, on the basis of their histological appearance, these appear to be ductal in origin. However, sequential analysis, by electron microscopy, of the development of pancreatic neoplasia in the hamster model indicates that acinar cells may participate in the histogenesis of "ductal" adenomas and carcinomas. Acinar cells appear to undergo changes in differentiation, including pseudoductular transformation, giving rise to a new population of cells that resemble ductular or centroacinar types. This new population may then proliferate to form, first, cystic foci and subsequently cystadenomas and adenocarcinomas. Mucous metaplasia appears to develop at late stages of tumor development. Although the participation of ductular and centroacinar cells in pancreatic carcinogenesis cannot be excluded, very few tumors arise from the ductal epithelium. It is possible that some human pancreatic adenocarcinomas may also have their origin from dysplastic acinar cells, by analogy with the hamster model: focal acinar dysplasia being common in human pancreatic cancer patients.

Effects of inhibiting hepatocarcinogenesis upon the early fine structural changes induced in rat hepatocytes by 3'-methyl-4-dimethylaminoazobenzene.

Young adult male Leeds strain rats were fed for up to 10 weeks on diets containing either 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) or 0.06% 3'-MeDAB together with 0.0067% 3-methylcholanthrene (MeCh), which is known to inhibit the induction of hepatocarcinogenesis by the azo dye. Controls received either MeCh alone or a normal diet. Animals were killed at 10 days, 4 weeks and 10 weeks and their liver tissues examined by electron microscopy. Although MeCh did not prevent the induction of some non-specific fine structural changes such as glycogen depletion and proliferation of the smooth endoplasmic reticulum (ER), it did prevent the early dislocation and dispersal of the rough ER that was elicited by 3'-MeDAB. This provides further evidence indicating the importance to liver tumorigenesis of this rough ER change, which appears to be induced by all classes of chemical hepatocarcinogens and which, in comparative studies with carcinogen/non-carcinogen pairs of related chemicals, also appears to be specific to carcinogens.

An electron microscope study of hepatocellular changes in the rat during chronic treatment with acetamide. Parenchyma, foci and neoplasms.

Male Leeds strain rats were fed a diet containing 5.0% by weight of acetamide (AA), for up to 35 weeks, inducing a high incidence of hepatic cell neoplasms. The sequential morphological changes induced by AA in the hepatic parenchymal cells were studied by electron microscopy and were compared with those observed in foci of cellular alteration, neoplastic nodules and hepatocellular carcinomata. The observed fine structural changes including early glycogen depletion, dispersal and dislocation of the rough endoplasmic reticulum (ER), smooth ER proliferation and nuclear irregularity and nucleolar abnormalities. Glycogenosis developed in some cells during treatment and was characteristic of the 'clear cell' foci, as well as being common in the neoplasms. The most consistent alteration, observed in parenchymal cells and the cells of foci, nodules and carcinomas, was that involving the rough ER. This persisted after withdrawal of AA from the diet, whereas many other changes were transient, and may represent a change in differentiation associated with neoplastic induction.

Induction of liver cell tumours in IF mice by paracetamol.

Groups of male and female IF strain mice were fed a diet containing either 0.5% or 1.0% paracetamol for up to 18 months. Among male mice fed the higher dose, the total liver cell tumour incidence was 87%, 21.7% developing hepatocellular carcinomas: both yields were statistically significant. The corresponding incidence in high dose females was 19.2% and 4.3%, respectively, only the former being significant. Foci of cellular alteration were also present in the livers of high dose mice of both sexes and also in those of the low dose males.

3-Methylcholanthrene-inhibition of hepatocarcinogenesis in the rat due to 3'-methyl-4-dimethylaminoazobenzene or 2-acetylamino-fluorene: a comparative study.

Male Leeds strain rats were given a diet containing either 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) or 0.05% 2-acetylaminofluorene (2-AAF), alone or together with 0.0067% 3-methylcholanthrene (MeCh). MeCh both reduced the incidence of hepatic neoplasms induced by 3'-MeDAB and delayed their development. With 2-AAF, however, inhibition of hepatocarcinogenesis by MeCh only appeared to operate during the period of treatment; thereafter the incidence of tumours rose rapidly and this was accentuated if dietary MeCh continued to be administered after the period of combined 2-AAF and MeCh treatment, suggesting a secondary, promoting effect of MeCH. In contrast, the inhibition by MeCh of carcinogenesis of Zymbal's gland due to 2-AAF was almost complete. Thus, in the long term MeCh has different effects upon 3'-MeDAB and 2-AAF carcinogenesis in the rat liver.

Ultrastructural analysis of pancreatic carcinogenesis. VI. Early changes in hamster acinar cells induced by N-nitroso-bis(2-hydroxypropyl)amine.

Male Syrian golden hamsters were given weekly s.c. injections of 250 mg/kg body weight of N-nitroso-bis(2-hydroxypropyl)amine (BHP) for up to 15 weeks. Electron microscope studies were carried out on early changes in the exocrine pancreas of these hamsters for 2 weeks to 15 weeks. The majority of observed alterations occurred in the acinar cells and included the appearance of "dark" and "light" cells, the former showing nuclear shrinkage and irregularity but maintaining a normal rough endoplasmic reticulum (ER) and zymogen content. The "light" cells exhibited a variety of early fine structural alterations including conformational changes in their rough ER, together with a reduction in zymogen granules, increased autophagic vacuoles and Golgi hypertrophy. Ducts and ductules were relatively unaffected. The observations indicate that the acinar cells were most affected, morphologically, by BHP and are consistent with the view that these cells are the primary target for BHP.

Ultrastructural analysis of pancreatic carcinogenesis. V. Changes in differentiation of acinar cells during chronic treatment with N-nitrosobis(2-hydroxypropyl)amine.

An electron microscope study was carried out on changes in the pancreatic acinar tissues of male Syrian hamsters during the course of induction of pancreatic neoplasia by lifetime weekly injections of N-nitroso-bis(2-hydroxypropyl)amine (BHP). The dominant process observed was a loss of differentiation of the acinar cells, leading to the development of a new population of cells that closely resemble centro-acinar or ductular cells. During this process, the original centro-acinar and ductular cells appear to be relatively unaffected by exposure to BHP. Thus, acinar cell de-differentiation appears, in this experimental model, to be an important preliminary stage in neoplastic development. It is argued that these findings may have implications for the histogenesis of human pancreatic adenocarcinoma, considered hitherto to be ductal in origin.