PubMed Health⌕ Search

Biomedical subjects

L K Keefer

Publications and source records attributed to L K Keefer.

At least 37 records · Page 2Linked to original sources
PubMed2000

Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release.

The preparation of hydrophobic polymer films (polyurethane and poly(vinyl chloride)) containing nitric oxide (NO)-releasing diazeniumdiolate functions is reported as a basis for improving the thromboresistivity of such polymeric materials for biomedical applications. Several different approaches for preparing NO-releasing polymer films are presented, including: (1) dispersion of diazeniumdiolate molecules within the polymer matrix; (2) covalent attachment of the diazeniumdiolate to the polymer backbone; and (3) ion-pairing of a diazeniumdiolated heparin species to form an organic soluble complex that can be blended into the polymer. Each approach is characterized in terms of NO release rates and in vitro biocompatibility. Results presented indicate that the polymer films prepared by each approach release NO for variable periods of time (10-72 h), although they differ in the mechanism, location and amount of NO released. In vitro platelet adhesion studies demonstrate that the localized NO release may prove to be an effective strategy for improving blood compatibility of polymer materials for a wide range of medical devices.

Animals↗
PubMed2000

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Antigens, Polyomavirus Transforming↗
PubMed1999

Pharmacokinetics and consistency of pericardial delivery directed to coronary arteries: direct comparison with endoluminal delivery.

BACKGROUND AND HYPOTHESIS: Pharmacologic modulation of the contents of the pericardial space has been shown to influence the response of coronary arteries to balloon injury. Endoluminal (EL) local delivery of various drugs into coronaries has been found to be limited by short residence time, as well as by highly variable deposited agent concentration. We hypothesized that compounds placed into the pericardial space (P) would penetrate into coronary tissue with greater consistency than seen after EL delivery and provide for prolonged coronary exposure to agents. METHODS AND RESULTS: 125I-labeled basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), albumin, or 131I-labeled diazeniumdiolated albumin (NONO-albumin) were delivered as model/therapeutic proteins into the porcine pericardial space (n = 15 pigs) or into coronaries using an EL delivery catheter (n = 48 arteries). In subjects receiving 125I-labeled proteins, the delivery target or mid-regions of the left anterior descending (LAD) and left circumflex (LCx) arteries were harvested at 1 h or 24 h for gamma-counting and autoradiography, and fractional intramural delivery (FID) or retention measured as percent agent in 100 mg artery/agent in infusate for both time points. In the animals receiving 131I-labeled NONO-albumin, serial gamma imaging was employed to evaluate the rate of redistribution in individual animals following either pericardial or endoluminal delivery. At 1 h, FID values ranged from 0.00064 to 0.0052% for P delivery (median 0.0022%), and from 0.00021 to 6.7 for EL delivery (median 0.27%). At 24 h, FID values ranged from 0.00011 to 0.003 for P delivery (median 0.0013), and from 0.0002 to 1.4 for EL delivery. The estimated T1/2 for bFGF redistribution from the vascular tissue was 22 h (P) and 7 h (EL), respectively, while the directly determined T1/2 values for NONO-albumin redistribution from the delivery region were 22.2 h (P) and 2.5 h (EL). CONCLUSIONS: These data show that pericardial fluid contents can access coronary arteries with intramural concentrations which typically vary by 10-15-fold, while EL delivery results in a remarkably wide intramural concentration range with up to 33,000-fold variability. The apparent redistribution rate is more rapid following EL delivery, possibly due to sustained diffusive tissue loading from the pericardial space. Pericardial delivery appears to offer substantial advantages over EL administration with respect to residence time and reproducibility.

Albumins↗
PubMed1999

Induction of penile erection by intracavernosal and transurethral administration of novel nitric oxide donors in the cat.

PURPOSE: The effects of novel nitric oxide (NO) donors administered intracavernosally and transurethrally on erectile function in the anesthetized cat were evaluated. MATERIALS AND METHODS: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal and transurethral injections of novel NO donors (MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO). All parameters were measured after administration of NO donors intracavernosally via a 30-gauge needle and urethrally via a Jelco i.v. catheter in a volume of 200 microliters. Systemic arterial pressure was also assessed in these experiments. All NO donors were compared with a triple-drug control combination comprised of papaverine (1.65 mg.), prostaglandin E1 (0.5 microgram.), and phentolamine (25 micrograms.). RESULTS: MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZI/NO and PROLI/NO induced dose dependent increases in intracavernosal pressure and penile length (p < 0.05) when administered intracavernosally. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. The maximum increase in cavernosal pressure in response to PROLI/NO and PAPA/NO was associated with no significant change in systemic arterial pressure. Transurethral administration of PROLI/NO and PIPERAZI/NO induced dose-dependent increases in cavernosal pressure and penile length (p < 0.05). The response was similar to that of the triple-drug control combination, except that transurethral PROLI/NO and PIPERAZI/NO had no significant effect on systemic blood pressure. CONCLUSIONS: NO donors caused dose-dependent increases in cavernosal pressure when administered intracavernosally and transurethrally. These data suggest further exploration of the use of NO donors for the treatment of erectile dysfunction.

Animals↗
PubMed1998

Involvement of K+ATP channels in nitric oxide-induced inhibition of spontaneous contractile activity of the nonpregnant human myometrium.

Nitric oxide (NO), an important endogenous substance, is known to be a strong relaxant of smooth muscle, including myometrium. It has been postulated that the relaxing effect of NO on smooth muscle is achieved by the stimulation of soluble guanylyl cyclase, which leads to an increase in the cyclic guanosine 3',5'-monophosphate (cGMP) levels and hyperpolarization of the cellular membrane. The aim of our study was to investigate the involvement of K+ATP channels in the mechanism of cGMP-independent nitric oxide-induced inhibition of contractile activity of the nonpregnant human myometrium, obtained at hysterectomy. Nitric oxide's influence on contractile activity was recorded in the presence of methylene blue and glybenclamide, blockers of soluble guanylyl cyclase and K+ATP channels, respectively. Nitric oxide, generated by the NO donor DEA/NO, caused a dose-dependent inhibition of the spontaneous contractile activity of human nonpregnant myometrium. Preincubation with methylene blue (5 microM) did not prevent NO-induced relaxation of uterine strips, while 1.5 microM glybenclamide blocked this effect. Our results indicate that nitric oxide relaxes human non-pregnant uterus through K+ATP channels, independent of the cGMP pathway.

Adult↗
PubMed1997

Targeting nitric oxide (NO) delivery in vivo. Design of a liver-selective NO donor prodrug that blocks tumor necrosis factor-alpha-induced apoptosis and toxicity in the liver.

We have designed a drug that protects the liver from apoptotic cell death by organ-selective pharmacological generation of the bioregulatory agent, nitric oxide (NO). The discovery strategy involved three steps: identifying a diazeniumdiolate ion (R2N[N(O)NO]-, where R2N = pyrrolidinyl) that spontaneously decomposes to NO with a very short half-life (3 s) at physiological pH; converting this ion to a series of potential prodrug derivatives by covalent attachment of protecting groups that we postulated might be rapidly removed by enzymes prevalent in the liver; and screening the prodrug candidates in vitro and in vivo to select a lead and to confirm the desired activity. Of five cell types examined, only cultured hepatocytes metabolized O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) to NO, triggering cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and protecting the hepatocytes from apoptotic cell death induced by treatment with tumor necrosis factor-alpha (TNF alpha) plus actinomycin D. In vivo, V-PYRRO/NO increased liver cGMP levels while minimally affecting systemic hemodynamics, protecting rats dosed with TNF alpha plus galactosamine from apoptosis and hepatotoxicity. The results illustrate the potential utility of diazeniumdiolates for targeting NO delivery in vivo and suggest a possible therapeutic strategy for hepatic disorders such as fulminant liver failure.

Animals↗
PubMed1997

Boundary layer infusion of nitric oxide reduces early smooth muscle cell proliferation in the endarterectomized canine artery.

To evaluate the direct effect of nitric oxide (NO) on vascular smooth muscle cell (SMC) proliferation in vivo, we used an expanded polytetrafluoroethylene (ePTFE)-based local infusion device to deliver an NO donor, proline/NO (PROLI/NO), to the luminal boundary layer of endarterectomized artery and the distal anastomosis of the graft in a canine model. Once delivered to the blood, PROLI/NO releases NO by a mechanism involving pH-dependent decomposition. Six dogs underwent bilateral femoral artery endarterectomies. ePTFE infusion devices, blindly primed with PROLI/NO to one artery or proline to the contralateral vessel, were anastomosed proximal to the injured segments so that each animal served as its own control. PROLI/NO or proline was continuously delivered for 7 days from an osmotic reservoir, through the wall of the graft infusion device. Euthanasia was carried out at 7 days, and the processed specimens were blindly analyzed for SMC proliferation at both graft anastomoses and endarterectomized segments by a bromodeoxyuridine index assay. All dogs survived with no clinical side effects. In comparing the treated and control vessels, NO released from PROLI/NO significantly reduced SMC proliferation by 43% (13.24 +/- 1.24% versus 23.24 +/- 1.01%, P = 0.004) at the distal anastomoses and by 68% (10.58 +/- 1.63% versus 25.17 +/- 3.39%, P = 0.007) at endarterectomized segments. However, there was no significant difference in blood flow measurements between treated and control arteries (56.25 +/- 6.50 ml/min versus 46.50 +/- 3.20 ml/min, P = 0.094). These data demonstrate that local boundary layer infusion of NO released from PROLI/NO significantly reduces SMC proliferation in injured arteries with no effect on regional blood flow. This study suggests a new strategy to inhibit early SMC proliferation in injured arteries and probably to control intimal hyperplastic lesion formation in the manipulated vessels.

Anastomosis, Surgical↗
PubMed1997

Quantification of diazeniumdiolate mutagenicity in four different in vitro assays.

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER/NO ([H2N(CH2)3NH(CH2)4N[N(O)NO-](CH2)3 NH3+]), which generate NO spontaneously with half-lives at 37 degrees C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/-), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the Ames Salmonella plate assay. In the experiments reported here, Salmonella typhimurium strain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mM (3.5 x control) and greater, while SPER/NO was mutagenic at 0.5 mM (2.7 x control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mM, inducing a mutant frequency per survivor that was 2.5 x control, while SPER/NO was mutagenic at 0.5 mM with a mutant frequency of 2.5 x control. When the CHO cells were given 10 repetitive 20 mM DEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 x control. In the L5178Y mouse lymphoma cell TK+/- assay, DEA/NO doubled the mutation rate at 1.82 mM, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mM. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mM that was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mM. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.

Animals↗
PubMed1997

Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia.

The roles of nitric oxide derived from either the constitutive endothelial NO synthase (eNOS or NOS3) or the inducible NOS (iNOS or NOS2) in hepatic injury during endotoxemia remain controversial. To investigate this further, rats received a bolus of lipopolysaccharide (LPS) following implantation of osmotic pumps containing one of two nonselective NOS inhibitors (NMA or NAME), one of two inducible NOS inhibitors (NIL or AG), or saline. The inhibitors were infused continuously into the liver via the portal vein. Treatment of LPS-injected rats with NMA and NAME resulted in 106 and 227% increases, respectively, in circulating hepatic enzyme levels compared to LPS-treated control rats. In contrast, infusion of the iNOS-selective inhibitors had no effect on the LPS-induced hepatic necrosis. In rats receiving NAME, LPS induced greater neutrophil infiltration and ICAM-1 expression than in the LPS + saline group, whereas NIL infusion did not. The increased hepatic necrosis and PMN infiltration in the LPS + NAME group was partially prevented by a simultaneous infusion of a liver-selective NO donor. Inhibition of PMN accumulation using an anti-ICAM-1 antibody or by PMN depletion using vinblastine pretreatment, however, did not reverse the increased necrosis with NAME infusion during endotoxemia. In contrast to the assessment for necrosis, increased apoptosis was observed in the livers of LPS-treated rats receiving infusions of either NAME or NIL, but not with LPS alone. These data indicate that NO produced by eNOS may be adequate to prevent necrosis by a mechanism independent of PMN, while induced NO appears to prevent apoptosis.

Animals↗
PubMed1997

Relative mutagenicities of gaseous nitrogen oxides in the supF gene of pSP189.

Gaseous nitric oxide (NO), an environmental pollutant found in cigarette smoke and diesel exhaust, has been shown to generate mutations in aerobic in vitro assays. The objective of this study was to identify which oxides of nitrogen, formed in the gaseous phase from NO, possess mutagenic activity. Samples of the plasmid pSP189, in 1 M HEPES buffer, pH 7.4, were exposed to preparations of nitrogen dioxide (NO2), dinitrogen trioxide (N2O3) or an air control. The gas mixtures were formed in a gas-tight syringe and were then introduced into 1 l flasks. The plasmid solution was introduced immediately afterwards. Transformation of Escherichia coli strain MBM7070 with the treated plasmids allowed analysis of mutation frequencies and the types of mutations induced in the target supF gene. The mutation frequency resulting from NO2 exposure was not different from that of the control. However, N2O3 produced a substantial number of mutations. The mutation frequency and the types of mutations were found to depend on the length of time that the gases were allowed to incubate in the syringe prior to introduction into the 1 l flasks (mutation frequency was maximal at approximately 2 min). The most prevalent mutations were AT-->GC transitions (68%), followed by GC-->AT transitions (30%), similar to previous findings when pure NO was bubbled through pSP189 solutions. These results suggest that it is N2O3, rather than NO2, that is the most likely source of mutagenic potential from gaseous nitrogen oxides.

Base Sequence↗
PubMed1996

Localizing antithrombotic and vasodilatory activity with a novel, ultrafast nitric oxide donor.

Reaction of nitric oxide (NO) with L-proline in methanolic sodium methoxide yields a diazeniumdiolate product, C5H7N3O4Na2.CH3OH (PROLI/NO), that can be stabilized in basic solution but that dissociates to proline (1 mol) and NO (2 mol) with a half-life of only 1.8 s at pH 7.4 and 37 degrees C. This kinetic behavior has allowed the generation of highly localized antiplatelet and vasodilatory effects. By infusing solutions containing 4 microM PROLI/NO in 0.1 M sodium hydroxide at the rate of 1 nmol.min-1 immediately upstream from a polyester vascular graft in the unheparinized baboon circulatory system, for example, platelet deposition at the normally thrombogenic graft surface was substantially reduced relative to controls receiving only 0.1 M sodium hydroxide. In a second study, infusion of PROLI/NO into the right atrium of sheep with induced pulmonary hypertension selectively dilalated the lung vasculature, dose-dependently reducing the pulmonary arterial pressure by as much as 9 mmHg with no observable effect on the systemic arterial pressure at an infusion rate of up to 24 nmol.kg-1.min-1. PROLI/NO could also be formulated as an insoluble polymer blend that released NO smoothly for prolonged periods. The results suggest that localized delivery of diazeniumdiolates such as PROLI/NO which generate NO with extreme rapidity on entering the blood stream may hold considerable promise for inhibition of thrombus formation, selective dilation of the vasculature, and other research and clinical applications.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

Nitric oxide-releasing polymers containing the [N(O)NO]- group.

Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

Animals↗
PubMed1996

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules.

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mM), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 microM), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of L-arginine was reversed by administration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 microM), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3',5'-cyclic monophosphate (8-B-cGMP) (1 mM), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETA/NO) and endogenously generated (L-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.

Aminoquinolines↗
PubMed1996

An evaluation of the roles of metabolic denitrosation and alpha-hydroxylation in the hepatotoxicity of N-Nitrosodimethylamine.

N-Nitrosodimethylamine (NDMA) is a potent hepatotoxic agent in rats. NDMA causes cell death that does not correlate with known mechanisms of toxicity such as the production of oxidative stress or covalent binding to proteins. The following studies were designed to determine whether NDMA cytotoxicity is the result of metabolic denitrosation or alpha-hydroxylation of the nitrosamine. We determined the toxicity of various metabolites of NDMA in monolayer cultures of primary rat hepatocytes. NDMA was toxic at 0.1 mM in our cultures, but the metabolites formaldehyde, methanol, and methylamine were not toxic at this concentration. We used diazeniumdiolates that spontaneously release nitric oxide (NO) in aqueous media to deliver NO to hepatocytes in culture. The results show that, while NO released from diazeniumdiolates causes death in hepatocytes, the levels of NO produced during NDMA metabolism are insufficient to account for the toxicity of the nitrosamine. NDMA-d6, the fully deuteriated form of NDMA that undergoes approximately twice as much denitrosation in vivo as NDMA, was significantly less cytotoxic than NDMA. In contrast, N-nitroso(acetoxymethyl)methylamine (AcO-NDMA), a stable precursor of the methanediazonium ion, was found to cause toxicity equivalent of NDMA on a molar basis. Altogether, our results with methylamine, formaldehyde, methanol, the diazeniumdiolates, and NDMA-d6 indicate that NDMA toxicity is not the result of metabolic denitrosation, while the toxicity of AcO-NDMA provides strong strong evidence that the formation of the methanediazonium ion via alpha-hydroxylation leads to cell death.

Animals↗
PubMed1996

Histogenesis and the role of p53 and K-ras mutations in hepatocarcinogenesis by glyceryl trinitrate (nitroglycerin) in male F344 rats.

Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding. The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development. Male F344 rats received a single i.g. intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e. for up to 78 weeks. Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age. Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed. No liver tumors developed in control rats or in rats that received GTN only by a single i.g. intubation, even when intubation was followed by PH. Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet. Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age. Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions. HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN. Incidence of HCC in the latter animals was 50-75%. Most HCCs were well differentiated. The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH. No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements. These were first or second position G-->T transversions or second position G-->A transitions. While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e. genetic and/or epigenetic) factors in carcinogenesis by GTN.

Animals↗