Biomedical subjects
J M Ward
Publications and source records attributed to J M Ward.
Hepatic lesions in Danish epileptics after long-term exposure to anticonvulsant drugs including phenobarbitone.
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Induction of hepatic metallothionein in male B6C3F1 mice exposed to hepatic tumor promoters: effects of phenobarbital, acetaminophen, sodium barbital, and di(2-ethylhexyl) phthalate.
The effects of various compounds known to be hepatic tumor promoters and toxins in the male B6C3F1 mouse liver, including di(2-ethylhexyl)phthalate (DEHP), acetaminophen (ACT), barbital (BB), and phenobarbital (PB) on hepatic metallothionein (MT) concentrations were assessed after chronic exposure. From 6 weeks of age, male mice were maintained on diets containing DEHP at 12,000 or 6000 ppm, ACT at 10,000 or 5000 ppm, BB at 1,000 ppm, or drinking water with PB at 500 ppm for up to 24 weeks. MT was measured in hepatic cytosol at 0, 2, 8, and 24 weeks of exposure. DEHP proved a very effective inducer, producing elevations of MT as high as 11-fold. The increases in hepatic MT with DEHP were both dose- and time-related. ACT was likewise effective in producing hepatic MT elevations (maximum 6.7-fold) in a dose- and time-related fashion. BB and PB, however, had no effect on hepatic MT levels at any time point. While DEHP, BB, and PB treatments produced hepatomegaly, histopathological analysis at 24 weeks revealed that in both DEHP- and ACT-treated livers hepatocellular proliferation was prominent while livers exposed to BB or PB showed predominantly hepatocellular hypertrophy. Gel-filtration of DEHP-treated liver cytosol revealed that zinc was associated with the MT peak. This peak also bound cadmium in vitro and could be extracted by heat treatment and selective acetone precipitation, both typical characteristics of MT. Further confirmation of the presence of MT after DEHP treatment was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% acrylamide). Results indicate that some, but not all, tumor promoters can induce target organ MT and that such an induction appears associated with those promoters inducing persistent cellular hyperplasia but not those inducing cellular hypertrophy.
Effects of sodium salts of phenobarbital and barbital on development of bladder tumors in male F344/NCr rats pretreated with either N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide or N-nitrosobutyl-4-hydroxybutylamine.
Promoting effects of sodium salts of phenobarbital (NaPB) and barbital (NaBB) on the development of bladder tumors were investigated in F344 male rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-nitrosobutyl-4-hydroxybutylamine (BBN). To initiate with FANFT, rats were fed 0.2% FANFT mixed in diet for either 2 or 6 weeks and 2 weeks later were offered diet containing 1000 ppm of NaPB or NaBB. Rats were killed either at 52 or 68 weeks of age. To initiate with BBN, rats were given 0.05% BBN in drinking water for 4 weeks and beginning 1 day later were fed NaBB mixed in diet at 1000 ppm for up to 52 weeks. NaBB promoted bladder carcinogenesis initiated by either FANFT or BBN; the incidence and average number of simple or preneoplastic nodular (PN) hyperplasias, papillomas, and carcinomas per 10 cm of urothelium were significantly increased in the groups receiving NaBB following exposure to FANFT for 6 weeks (p less than 0.05) or BBN for 4 weeks (p less than 0.01). No such effect was seen in rats fed FANFT for only 2 weeks. NaPB also significantly increased (p less than 0.05) the frequency of preneoplastic PN hyperplasias but not the average number of papillomas and carcinomas per 10 cm of urothelium in rats fed FANFT for 6 weeks. NaBB was an effective promoter of bladder carcinogenesis under these experimental conditions, as expected from its known promoting effect on transitional epithelium of the renal pelvis, but NaPB in contrast did not affect the incidence or multiplicity of bladder papillomas or carcinomas under these conditions. NaPB could be considered a promoter for bladder urothelium only by the less rigorous criterion that it increased the frequency of preneoplastic PN hyperplasia.
Hepatotoxicity of agents that enhance formation of focal hepatocellular proliferative lesions (putative preneoplastic foci) in a rapid rat liver bioassay.
The histopathology of hepatic toxicity for 58 chemicals previously tested in a rapid rat liver bioassay for demonstrating potential hepatocellular carcinogens and/or tumor promoters was reviewed. Rats received the test diet for 1 week prior to partial hepatectomy and for an additional 5 weeks thereafter at doses near the estimated maximally tolerated dose. These rats served as controls for others receiving initiation by N-nitrosodiethylamine (DEN) and the test diets. Twenty-two of these chemicals were previously found to enhance the formation of glutathione S-transferase, placental form (GST-P)-positive putative preneoplastic hepatocellular foci (promoters) following DEN initiation in this rapid bioassay, whereas 36 chemicals did not. Of the agents that promoted GST-P-positive foci, 14/22 (63.6%) produced toxic hepatocyte lesions while only 4/36 (11.1%) of the nonpromoters did so at the doses used. Biliary toxicity was found for 7/22 (31.8%) of the promoters and 6/36 (16.7%) of the nonpromoters. Only 2/13 (15%) chemicals that inhibited GST-P-positive foci produced hepatic toxicity. Thus, agents that were presumed hepatic tumor promoters characteristically were hepatotoxins while nonpromoters of carcinogenesis were not hepatotoxins in this rapid rat liver bioassay.
Barbital sodium, a tumor promoter for kidney tubules, urinary bladder, and liver of the F344 rat, induces persistent increases in levels of DNA synthesis in renal tubules but not in urinary bladder epithelium or hepatocytes.
The nephrotoxicity of barbital sodium (NaBB), a renal and urinary bladder tumor promoter, was investigated in male F344/NCr rats. In an 8-week toxicity study, NaBB was administered to 6-week-old male rats for 8 weeks at dietary levels of 16,000, 8000, 4000, 2000, 1000, or 0 ppm. Rats tolerated NaBB at levels of 4000 ppm and below with no weight depression or mortality. Liver-to-body weight ratios, however, were significantly increased at 4000 ppm, and toxic renal lesions were observed histologically. Light microscopic studies of male rats fed 1000 ppm NaBB for 2-52 weeks or 4000 or 8000 ppm for 8 weeks revealed elevated levels of DNA synthesis in renal tubular cells as measured with tritiated thymidine autoradiography or 5-bromo-2'-deoxyuridine immunohistochemistry that were associated with degenerative and regenerative nephrotoxic lesions. Increased labeling indices of urothelium and hepatocytes were not seen in rats exposed to 1000 ppm NaBB which is effective as a bladder and liver tumor promoter at these doses. These studies provide evidence for the chronic nephrotoxicity and renal tubular hyperplasia induced by NaBB in F344 rats, which are associated with the tumor-promoting activity of NaBB at the doses studied. Hyperplasia in the urinary bladder or liver was not found, however, for this bladder and liver tumor promoter. The conflicting findings in liver, bladder, and kidney are discussed.
Tumor promoting activities of ethylphenylacetylurea and diethylacetylurea, the ring hydrolysis products of barbiturate tumor promoters phenobarbital and barbital, in rat liver and kidney initiated by N-nitrosodiethylamine.
The ring hydrolysis products of the multi-tissue tumor promoting barbiturates, phenobarbital (PB) and barbital (BB), were fed to F344/NCr male rats previously given a single initiating injection of N-nitrosodiethylamine. Ethylphenylacetylurea (EPAU) and diethylacetylurea (EEAU), derived, respectively, from PB and BB, both promoted development of hepatocellular adenomas, but were much weaker in this respect than PB. Both acetylureas were also selective inducers of P-450IIB1-mediated benzyloxyresorufin O-dealkylase activity, but both were much less potent inducers than PB. Neither EPAU nor EEAU had an effect on tumor development in the thyroid, unlike both PB and, as shown previously, BB. EEAU, but not EPAU, strongly promoted development of renal cortical epithelial tumors. The opening of the barbiturate heterocyclic ring and the subsequent decarboxylation to yield the dialkylacetylurea analogs thus resulted in compounds displaying a marked reduction in liver tumor promoting activity. EEAU appears to possess an ability to promote renal neoplasms similar to that of its parent compound, BB. Ring opening appears to be accompanied by loss of promoting activity for thyroid follicular epithelium.
Promotion of malignant 'embryonal' liver tumors by phenobarbital: increased incidence and shortened latency of hepatoblastomas in (DBA/2 x C57BL/6)F1 mice initiated with N-nitrosodiethylamine.
In order to analyze the genetics of susceptibility to promotion of hepatocarcinogenesis in DBA/2NCr and C57BL/6NCr mice by phenobarbital (PB), reciprocal F1 hybrid male mice were given 90 mg N-nitrosodiethylamine (NDEA)/kg body weight, i.p. at 5 weeks of age followed by 0.05% PB in drinking water. Hepatocellular adenomas and carcinomas were comparably increased in incidence and multiplicity in both reciprocal hybrids over mice given NDEA alone. Eight of 10 D2B6F1 progeny of DBA/2NCr females mated with C57BL/6NCr males, but only 1/10 B6D2F1 mice (progeny of C57BL/6NCr females mated with DBA/2NCr males) given PB after NDEA initiation developed single or multiple hepatoblastomas within 42 weeks. These small-celled, intensely basophilic tumors were characteristically hemorrhagic and highly malignant. Hepatoblastomas were mostly found within or adjacent to hepatocellular tumors. No hepatoblastomas were seen in either hybrid given NDEA alone. PB consistently enhanced development of malignant hepatoblastomas, as well as promoted hepatocarcinogenesis in D2B6F1 males, but did not elicit hepatoblastoma development in B6D2F1 males that were genetically identical to D2B6F1 males except for the reverse origin of their X and Y chromosomes.
Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes.
The tumor-promoting ability of clonazepam (CZP), a widely used benzodiazepine anticonvulsant, was investigated in an in vivo mouse liver tumor promotion assay and an in vitro mouse hepatocyte intercellular communication assay. The development of preneoplastic hepatocellular foci of cellular alteration and hepatocellular neoplasms was studied in male B6C3F1 mice initiated, at 5 weeks of age, with a single i.p. injection of N-nitrosodiethylamine (NDEA; 90 mg/kg body weight) in tricaprylin, followed by administration of either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%) in diet beginning 2 weeks after carcinogen injection and continuing to 60 weeks of age. Several mice from each group were killed after 9, 21, 33 or 53 weeks on test diet, and portions of liver and other organs were fixed in formalin and examined histologically. Unlike PB, CZP did not promote the development of preneoplastic hepatocellular foci or neoplasms (adenomas and carcinomas) in NDEA-initiated mice. Following limited (2 weeks) dietary exposure at 0.15%, CZP was a potent inducer of hepatic P450IIB1-mediated alkoxyresorufin O-dealkylase activities. In contrast, the degree of induction in hepatic tissue from mice fed 0.136% CZP for 53 weeks was markedly lower than that in mice fed 0.05% PB for 53 weeks. In the in vitro assay, diazepam, a strong tumor promoter in mouse liver, significantly inhibited mouse hepatocyte gap junctional intercellular communication, while CZP had no significant effect on this parameter. Thus, CZP, a drug structurally related to diazepam, is inactive as a liver tumor promoter in mice.
Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae.
The aminoglycoside phosphotransferase gene (aph) from the neomycin producer Streptomyces fradiae encodes an enzyme (APH) that phosphorylates, and thereby inactivates, the antibiotic neomycin. Two promoters were identified upstream of and oriented toward the aph coding sequence. One promoter (aphp1) initiated transcription at the A of the ATG translational initiation codon, or one to two bases upstream. Mutations made in this promoter region identified functionally important nucleotides and verified that the aphp1 transcript was translated to yield the APH protein, despite the lack of a conventional ribosome binding site. A second aph promoter, aphp2, initiated transcription 315 bp upstream of the translational initiation codon but gave transcripts that appeared to terminate before reaching the coding sequence. Multiple transcriptional initiation sites (pA1-pA5) were identified also in the aph regulatory region oriented in the opposite direction to aph transcription. Promoters for the pA2 and pA4 transcripts overlap with aphp1 such that down-promoter mutations in aphp1 also reduce transcription from the overlapping pA promoters.
The influence of parental attitude and behavior on early adolescent cigarette smoking.
In 1983, Nolte and colleagues reported parental attitude may be more powerful than parental behavior in shaping adolescent cigarette smoking behavior. This study replicates the finding of Nolte et al and suggests parents need to be actively recruited to discourage their children from smoking, regardless of their own behavior. Fewer parents actively discourage youth smoking today than in 1983, a possible unfortunate result from an apparently successful effort to change the public attitude toward cigarette smoking.
Immunohistochemical localization of cytochromes P450 with polyclonal and monoclonal antibodies.
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Quantitative analysis of alveolar type II cell tumors in mice by whole lung serial and step sections.
Alveolar type II cell tumors were induced transplacentally by intraperitoneal injection of pregnant C3H/HeNCr MTV- or Swiss Webster mice with N-nitrosoethylurea at a dose of 0.5 mmol/kg and 0.74 mmol/kg. At different time points after birth (1-32 weeks), the entire lungs from 40 of the male offspring were inflated with Bouin's fixative, separated into lobes, and sectioned at 5 microns serially to detect every microscopic lesion. Results were compared with those obtained from examining only every 10th, 20th, or a single midlevel section from the same material. On average, 150 serial sections were prepared per mouse lung. Initially, only purely solid/alveolar or purely tubulopapillary types were observed but with tumor progression, papillary structures developed within solid tumors resulting in mixed neoplasms. Analyzing mouse lungs in step sections of every 10th section (50-60 microns), 5/238 (2%) of the tumors were missed, in step sections of every 20th section (100-120 microns), 16/238 (7%) of the tumors were not detected and usually less than half of the tumors were seen in the single mid-level section. The approximate size of the neoplasms is indicated by the total number of sections per tumor. The dimensions of tumors evaluated with step sections of 10 or 20 were comparable to the size observed with serial sections. It is concluded that the evaluation of mouse lung tumors in steps of approximately 50 microns is basically equivalent to the study of serial sections and appears to be a feasible method to assess the complete incidence, histological type, and size of all proliferative processes throughout the entire lung.
Increased levels of DNA synthesis in hyperplastic renal tubules of aging nephropathy in female F344/NCr rats.
Histoautoradiography was done to evaluate cell turnover by measuring levels of DNA synthesis in the different anatomic areas of the kidney in aging (76 to 103 weeks of age) female F344/NCr rats after injection of tritiated thymidine (3H-thymidine). All areas showed increased 3H-thymidine labeling indices (LI), including cortical and medullary tubules and interstitium. The extent of increase was directly correlated with increasing severity of the nephropathy. Atrophic, degenerative, and hyperplastic cortical tubules in areas of nephropathy had LI eight to nine times greater than nonaffected areas and more normal kidneys. These observations suggest that the hyperplastic responses of renal tubules to the unknown etiologic agents of aging nephropathy may be similar to those of tubules responding to chemical toxins.
Immunohistochemical demonstration of Clara cell antigen in lung tumors of bronchiolar origin induced by N-nitrosodiethylamine in Syrian golden hamsters.
Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffusely that were immunoreactive for cytokeratin. Immunoreactivity for cytokeratin was also noted for hyperplastic bronchiolar neuroepithelial bodies. Cellular hyperplasias extending out into the alveolar parenchyma contained ciliated cells and frequently consisted of cells immunoreactive for CCA, showing them to be of bronchiolar Clara cell origin. Tumors developed from bronchiolar cell hyperplasias localized within bronchioles and from bronchiolar cells lining former alveolar walls. Neoplastic growth patterns were tubulo-papillary, forming loose networks or densely cellular areas. Immunoreactivity for cytoplasmic CCA was found in 50% of the tumors and was seen most frequently in small cuboidal cells and larger, vacuolated cells scattered throughout the neoplasms. In summary, evidence is presented that NDEA-induced pulmonary tumors of the Syrian golden hamster originated from cells lining bronchioles and from extrabronchiolar Clara cell hyperplasias of the terminal bronchioles. As the pulmonary tumors of the hamsters progressed towards a squamoid cell type, CCA was no longer detectable but cells became immunoreactive for keratin.
Immunohistochemistry of human immunodeficiency virus in the central nervous system and an hypothesis concerning the pathogenesis of AIDS meningoencephalomyelitis.
Early events of HIV infection of the CNS are not yet clear. HIV infection in most recent cases, generally shows a prolonged interval between diagnosis and death. HIV infection, months to years before the patient's death, may or may not result in early neurologic symptoms. AIDS patients with spinal cord symptoms often show a sudden onset of long tract signs and a temporally related altered mental status indicating the appearance of both myelitis and encephalitis. Immunohistochemical localization of the HIV cell-surface receptor protein, CD4, and of HIV antigens in cerebral and lymph node venular endothelial cells suggests that a natural occurrence or induction of CD4 protein in some endothelial cells allows transmission of HIV from circulating infected white blood cells preferentially to certain tissues through endothelial cell infection. HIV immunolocalization is present in perivascular astrocytes, particularly in long white matter tracts, and on the surface of oligodendrocytes. HIV immunoreactivity is mostly in macrophages and multinucleated cells in a typical autopsy case, but this may be due to the clearing of HIV antigen from early sites of infection by the hematogenous cells. Not all immunoreactivity for HIV antigens is necessarily due to HIV gene products. Cross reacting epitopes, such as that of HIV envelope glycoprotein gp120 and neuroleukin, may be "seen" not only by antibodies on tissue sections, but by an AIDS patient's immune system, thus targeting a CNS antigen for immune-complex formation. Evidence for hypersensitivity disease in the CNS in AIDS includes the frequent findings of demyelination, perivenous chronic inflammation, chronic vasculitis, and perivenous hemorrhages. The white matter demyelination so frequently reported in all areas of the CNS in AIDS could be the result of a combination of factors that include direct HIV vasculitis, opportunistic infections, and hypersensitivity responses. The blood-brain barrier is breached when immune-related antigens interact on CNS vascular endothelial cells. Perhaps the CD4 antigen, which responds to interaction with antigen-presenting cells and enhances cellular immune activity, is induced or increased in the CNS in association with immune activity and in the presence of a leaky blood-brain barrier. Therefore, with or without HIV in the CNS, hypersensitivity disease, including demyelination, may be the result of long-standing activity of the immune system in AIDS patients.
Comparative immunohistochemical reactivity of monoclonal and polyclonal antibodies to H-ras p21 in normal and neoplastic tissues of rodents and humans.
Five mouse monoclonal antibodies (mES 2, 4, 8, 13 and 20) produced against bacterially expressed BALB ras p21 and five other monoclonal or polyclonal antibodies (Cetus rabbit or mouse pan p21, RAP-5, Triton Ha-ras, Y13-259) to H-ras p21 were used for comparative immunohistochemical detection of H-ras p21 by the avidin biotin peroxidase-complex technique in selected normal fixed tissues and tumors from rats, mice, and humans. Nine of these antibodies were immunoreactive with cell membranes and cytoplasm of harvey sarcoma virus-induced sarcoma cells, but usually only with Bouin's fixed tumors. A few normal tissues were immunoreactive with some of the antibodies except for many immunoreactive tissues found with mES 13. Although mES 13 stained sarcoma cells on the cell membrane, a prominent granular staining, which appeared to be mitochondrial or lysosomal, was seen in many normal and neoplastic rodent tissues and in normal human colon, colon polyps, and carcinomas. Interpretation of positive immunoreactivity with any antibody was sometimes difficult due to nonspecific (background) staining. The cellular pattern of specific reactivity (membrane or granular) and inhibition of immunoreactivity by absorption of the antisera with H-ras p21 was therefore important. Western blots with BALB transforming (Lys 12) p21 expressed in E. coli revealed reactivity of all antibodies except for RAP-5. In addition, immunoblots of solubilized proteins from the EJ cell line with RAP-5 indicated reactivity of this monoclonal antibody with proteins of several different molecular weights. RAP-5 also never specifically immunoreacted with cell membranes of normal or malignant cells including EJ cells. Interpretation of these findings in comparison with those in published reports of H-ras p21 localization in fixed tissue sections is discussed, including the importance of fixative, specific antibody and controls.
P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats.
Male F344/NCr rats, 6 wk old, were fed 500 ppm of phenobarbital (PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or 5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufin O-dealkylase and aminopyrine N-demethylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective. To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 rats were given a single i.p. injection of 75 mg N-nitrosodiethylamine/kg body weight. Beginning 2 wk later, they were placed either on normal diet or diet containing 500 ppm of PB or equimolar doses of EPH or EEH for the remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificed at either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk and hepatocellular carcinomas at 78 wk in N-nitrosodiethylamine-initiated rats. Neither the incidence of hepatocellular neoplasms nor the number and size of hepatocellular foci was significantly increased by EEH. At 78 wk, both PB and EPH enhanced the development of thyroid follicular cell neoplasms in N-nitrosodiethylamine-initiated rats while no such enhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticonvulsant, like the structurally similar PB, promoted hepatocellular and thyroid follicular cell carcinogenesis and induced the PB-inducible form(s) of cytochrome P-450 (P-450b) in rats. In contrast, EEH unlike barbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochrome P-450 in rats.