Organ specific acute toxicity of the carcinogen trans-4-acetylaminostilbene is not correlated with macromolecular binding

trans-4-Acetylaminostilbene (trans-AAS) is acutely toxic in rats and lesions are produced specifically in the glandular stomach. Toxicity is slightly increased by pretreating the animals with phenobarbital (PB) and is completely prevented by pretreatment with methylcholanthrene (MC). The prostaglandin inhibitors, indomethacin and acetyl salicylic acid, do not reduce toxicity. The high efficiency of MC suggested that toxicity is caused by reactive metabolites. trans-[3H]-AAS was administered orally to untreated and to PB- or MC-pretreated female Wistar rats and target doses in different tissues were measured by means of covalent binding to proteins, RNA and DNA. Macromolecular binding in the target tissue of poisoned animals was significantly lower than in liver and kidney and comparable to other non-target tissues. Pretreatment with MC lowered macromolecular binding in all extrahepatic tissues but not in liver. These findings are not in line with tissue specific metabolic activation. The only unique property of the target tissue, glandular stomach, that we observed was a particular affinity for the systemically available parent compound. In the early phase of poisoning, tissue concentrations were exceedingly high and the stomach function was impaired.     

The parotid gland: a new target organ for vitamin D action.

Radioactively labelled cholecalciferol was administered continuously to rats which were fed a vitamin D-deficient diet. It has been possible to show that all the metabolites of the cholecalciferol which normally occur in known target tissues of vitamin D are present in the parotid gland, and the pattern resembled that obtained for the kidney, a known target tissue for vitamin D action. The accumulation of cholecalciferol metabolites in the parotid gland was shown to be functional, as a calcium-binding protein was found to be present in the gland, possessing similar properties to the renal vitamin D-dependent calcium-binding protein.     

The parotid gland: A new target organ for vitamin D action

Radioactively labelled cholecalciferol was administered continuously to rats which were fed a vitamin D-deficient diet. It has been possible to show that all the metabolites of cholecalciferol which normally occur in known target tissues of vitamin D are present in the parotid gland, and the pattern resembled that obtained for the kidney, a known target tissue for vitamin D action.The accumulation of cholecalciferol metabolites in the parotid gland was shown to be functional, as a calcium-binding protein was found to be present in the gland, possessing similar properties to the renal vitamin D- dependent calcium-binding protein.     

Genomic hypomethylation is specific for preneoplastic liver in folate/methyl deficient rats and does not occur in non-target tissues

Chronic dietary insufficiency of the lipotropic nutrients choline and methionine is hepatocarcinogenic in male rats and certain mouse strains. Despite the fact that DNA hypomethylation is a hallmark of most cancer genomes, the tissue-specific consequences of this alternation with respect to tumorigenesis remain to be determined. In the present study, the folate/methyl deficient model of multistage hepatocarcinogenesis was used to evaluate in vivo alterations in DNA methylation in the liver, the carcinogenesis target tissue, and in non-target tissues, including pancreas, spleen, kidney, and thymus, of male F344 rats. By utilizing the HpaII/MspI-based cytosine extension assay, we demonstrated that the percent of CpG sites that lost methyl groups on both strands progressively increased in liver tissue after 9, 18, and 36 weeks of folate/methyl deficiency. The endogenous activity of DNA methyltransferase in liver of rats fed with folate/methyl deficient diet for the 36-week period gradually increased with time. In contrast, non-target tissues displayed no changes in DNA methylation level or activity of DNA methyltransferase. The failure of DNA methyltransferase to restore and maintain DNA methylation patterns in preneoplastic liver tissue may lead to the establishment of tumor-specific DNA methylation and DNA methyltransferase profiles that are not expressed in normal liver. These results provide additional information about alterations in DNA methylation during early preneoplastic stages of carcinogenesis. They also demonstrate that DNA hypomethylation is localized to tissue that undergoes carcinogenesis, and is not altered in non-target tissues.     

Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.     

Oxysterols increase in diabetic rats

To address whether diabetes enhances lipid peroxidation and attenuates nitric oxide (NO) generation resulting in tissue complications, we measured oxysterols and NO metabolites (NOx) in the tissues of diabetic Wistar rats. After 4 weeks of streptozotocin injection (STZ, 80?mg/kg, i.p.), we measured 7α- and 7β-hydroperoxycholest-5-en-3β-ol (7α-OOH and 7β-OOH), 7α- and 7β-hydroxycholesterol (7α-OH and 7β-OH) and 7-ketocholesterol (7-keto) by HPLC in the kidneys, heart, and liver. All the oxysterols were much higher in the diabetic than in sham rats, while the extent of the increase was higher in the order of the kidney, heart, and liver. Together with high blood urea nitrogen, the data indicate that the kidney is the predominant target of early diabetic complications. Plasma NOx were decreased by 20% in the STZ rats. The enhanced oxidative stress in diabetes would increase oxysterols by peroxidation, while superoxide is known to reduce NO by reaction to form another potent oxidant peroxynitrite.     

Oxysterols increase in diabetic rats

To address whether diabetes enhances lipid peroxidation and attenuates nitric oxide (NO) generation resulting in tissue complications, we measured oxysterols and NO metabolites (NOx) in the tissues of diabetic Wistar rats. After 4 weeks of streptozotocin injection (STZ, 80 mg/kg, i.p.), we measured 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH), 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH) and 7-ketocholesterol (7-keto) by HPLC in the kidneys, heart, and liver. All the oxysterols were much higher in the diabetic than in sham rats, while the extent of the increase was higher in the order of the kidney, heart, and liver. Together with high blood urea nitrogen, the data indicate that the kidney is the predominant target of early diabetic complications. Plasma NOx were decreased by 20% in the STZ rats. The enhanced oxidative stress in diabetes would increase oxysterols by peroxidation, while superoxide is known to reduce NO by reaction to form another potent oxidant peroxynitrite.     

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.     

Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15).

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO₂ method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO₂ assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO₂ method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO₂ method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.     

Conservation of genomic imprinting at the NDN, MAGEL2 and MEST loci in pigs

Imprinted genes have important effects on the regulation of fetal growth, development, and postnatal behavior. However, the study of imprinted genes has been limited in mammalian species other than human and mouse. Therefore, the study of porcine imprinted genes is useful for defining the extent of conservation of genomic imprinting among different species. In this study, the imprinting status of porcine NDN, MAGEL2 and MEST genes was determined by direct sequencing of the cDNAs and detection of single nucleotide polymorphisms (SNPs) identified in individuals from reciprocal crosses between Meishan and Large White pigs for allele discrimination. The analysis was carried out in 13 different tissues (skeletal muscle, fat, pituitary gland, heart, lung, liver, kidney, spleen, stomach, small intestine, uterus, ovary and testis) from 12 two-month-old piglets. Imprinting analysis showed that NDN and MAGEL2 were paternally expressed in all tissues where the genes were expressed as in human and mouse. Interestingly, MEST showed tissue-specific imprinting, being paternally expressed in skeletal muscle, fat, pituitary gland, heart, kidney, lung, stomach and uterus, and maternally expressed in spleen and liver.     

Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke

Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.     

Immobilization stress in rat tissues: alterations in protein oxidation, lipid peroxidation and antioxidant defense system

We determined the effects of immobilization stress on antioxidant status, protein oxidation and lipid peroxidation in brain, liver, kidney, heart and stomach of rats. Sixteen male Wistar rats (3 months old) were divided into controls (C) and immobilization stress group (IS). IS rats were immobilized for 180 min/day for 15 days. Plasma corticosterone levels were increased in IS group. Copper,zinc-superoxide dismutase activities were increased in brain, liver and kidney, but decreased in the heart and stomach after immobilization. Catalase activities were increased in brain, kidney and heart, and decreased in liver and stomach. Selenium-dependent glutathione peroxidase activities were decreased in brain and kidney, but increased in heart and stomach. Reduced glutathione levels were decreased, while protein carbonyl, conjugated dienes and thiobarbituric acid-reactive substances levels were increased in all tissues. Our results showed that the response of antioxidant defense system to stress differs for each tissue, and protein oxidation and lipid peroxidation is induced by immobilization stress in peripheral tissues.     

Similarities between the biochemical actions of cycasin and dimethylnitrosamine

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.     

Cell type-specific expression of beta-carotene 15,15'-mono-oxygenase in human tissues.

We studied the cell type-specific expression of human beta-carotene 15,15'-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.     

Carcinoma and cirrhosis of the liver in A X C male rats with one kidney ingesting 0.025% N-2-fluorenyldiacetamide.

The role of the kidneys in hepatic carcinogenesis was studies in inbred A X C strain male rats ingesting 0.025% N-2-fluorenyldiacetamide. The experimental groups consisted of male rats with both kidney intact and male rats that had the left kidney removed. The incidence of hepatic carcinomas and the number of rats with large carcinomas, multiple carcinomas, poorly differentiated and undifferentiated carcinomas, and metastases was greater in rats with a left nephrectomy. The incidence of cirrhosis was the same in animals in both groups; however, cirrhosis was more severe in degree in the rats with the left kidney removed. Some animals in the latter group also developed carcinosarcomas of the salivary glands. The animals with one kidney apparently were not able to excrete the active metabolites of N-2-fluorenyldiacetamide as readily as the animals with both kidneys intact. The metabolites were then returned to the liver and salivary gland.     

Maternal-perinatal interrelationships of vitamin D metabolism in rats.

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Immunochemical characteristics of the peroxidases of several mouse tissues]

Enzymes catalyzing peroxidase reaction of a lysosomal fraction in bone marrow, leucocytes, spleen, thyroid gland, stomach, kidney, heart, lungs, brain and skeletal muscle of mice were investigated by immunochemical methods. A high level of peroxidase activity was discovered in leucocytes, bone marrow, spleen, heart and lung, a lower activity appeared to be characteristic of liver, thyroid gland and kidney. The peroxidase activities in brain, skeletal muscle and stomach were low. The reaction of immunoprecipitation with myeloperoxidase-specific antiserum revealed considerable antigenic distinctions between the enzymes catalysing peroxidase reaction in various tissues of mice.     

The distribution of radioactivity in rats given [3H]bishydroxymethyl-1-methylpyrrole and its relationship to that from [3H]synthanecine a bis-N-ethylcarbamate

The distribution of radioactivity was measured in rats various times after single intravenous doses of tritium-labelled 2,3-bis-hydroxymethyl-1-methylpyrrole (BHMP). Over half the dose was excreted in urine during the first day; less than a seventh this amount was found in the faeces. The level in glandular stomach was much higher than in any other organ and there was evidence that this was related to the acidity of this tissue. With this exception, the radioactivity in other tissues was lower than after an equivalent dose of tritiated synthanecine A bis-N-ethylcarbamate, and that in liver tissue was more easily solubilised than after the latter compound. The results indicate it is unlikely that more than a small amount of free BHMP is released into the bloodstreams of rats given synthanecine A bis-N-ethylcarbamate.When [³H]BHMP is given by stomach tube much radioactivity remains within the gut and there is no exceptional binding to glandular stomach tissue. Whereas the tissue binding as well as chemical and toxicological properties of BHMP are similar to those of dehydroretronecine, the binding properties of synthanecine A bis-N-ethylcarbamate are likely to resemble those of monocrotaline and similar pyrrolizidine alkaloids.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.