An evaluation of chronic 6- and 12-month rat toxicology studies as predictors of 2-year tumor outcome.

Chronic 6- and 12-month rat toxicology studies were evaluated for their ability to predict tumor outcome in 2-year rat carcinogenicity studies for 80 pharmaceuticals from commercial and Merck databases. The data consisted of 62 (6-month) and 54 (12-month) studies, which included 30 rat carcinogens and 50 noncarcinogens in 2-year studies. The histopathology findings considered as evidence of potential preneoplasia in the chronic studies were hyperplasia, cellular hypertrophy, and atypical cellular foci. The authors hypothesized that a whole animal-based approach should be taken, wherein (1) evidence of potential preneoplasia in any tissue may serve as a sensitive predictor of tumor outcome in any tissue in the whole animal and not necessarily the same tissue and (2) the absence of evidence for potential preneoplasia in all tissues may serve as a strong negative predictor of tumor outcome in any tissue. Based on this whole animal approach, 25 of 30 rat carcinogens showed histopathologic signals in chronic toxicology studies, yielding a test sensitivity of 83%. The negative predictivity of the absence of histopathology findings in chronic toxicology studies of 50 nontumorigenic compounds was 88%. The value of the extra 6-month treatment was not apparent. The 5 false negatives (negative chronic studies but positive 2-year studies) are for marketed drugs approved for non-life-threatening conditions and associated with rat-specific mechanisms. The absence of preneoplasia in the whole animal is a reliable predictor of negative tumor outcome in 2-year rat studies, and approximately 50% rat carcinogenicity studies could be eliminated for the 80 pharmaceuticals examined, with no risk to humans and with a substantial reduction in animal usage and drug development time.

Differential local and systemic regulation of the murine chemokines KC and MIP2.

We characterized the relative biological activity and expression of two murine chemokines that may serve as functional homologues for human IL-8, KC, and macrophage inflammatory protein 2 (MIP2). Recombinant chemokines were produced in bacterial expression systems and antibodies specific for KC or MIP2 were raised. In vitro assays showed that KC elicited 4-fold greater neutrophil chemotaxis compared with MIP2, while MIP2 elicited significantly greater release of elastase. Lipopolysaccharide- (LPS) stimulated macrophages (8 h) secreted more MIP2 (approximately 10 ng/mL) compared with KC (approximately 4 ng/ml) and expression of either murine chemokine was independent of TNFalpha or IL-1beta production. Thioglycollate (thio) and glycogen (gly) induced peritonitis produced more KC (thio = 7.1 and gly = 2.5 ng/mL) in the peritoneum compared with MIP2 (thio = 4.5 and gly = 0.3 ng/mL). Plasma KC levels were very high after either challenge (approximately 24 ng/mL), which was >50-fold more than the systemic increase in MIP2 (approximately 0.3 ng/mL). Our data demonstrate that while KC and MIP2 have similar in vitro production characteristics, KC appears to be a more potent and systemically distributed chemokine during acute in vivo inflammation, while MIP2 expression appears limited to localized expression.

Tumor necrosis factor alpha is not required for WY14,643-induced cell proliferation.

It has been proposed that the cytokine tumor necrosis factor alpha (TNFalpha) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNFalpha knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNFalpha knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNFalpha knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (approximately 8-fold) and mRNA content (approximately 5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (approximately 15-fold) in both wild-type C57Bl/6 and TNFalpha knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNFalpha mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNFalpha signaling.

Dexamethasone selectively inhibits WY14,643-induced cell proliferation and not peroxisome proliferation in mice.

It has been proposed that the hepatocellular proliferation induced by peroxisome proliferators may occur through an indirect mechanism involving cytokine release as opposed to direct regulation of cell growth genes by PPARalpha. We compared the induction of peroxisome proliferation and cell proliferation in C57Bl/6 mice treated with 100 mg/kg/day WY14,643 in the presence or absence of increasing doses of dexamethasone (DEX), an inhibitor of the release of proinflammatory cytokines. Biochemical markers of peroxisome proliferation, including fatty acyl-CoA oxidase activity, CYP4A content, and liver-to-body-weight ratios were markedly increased in the WY14,643-treated mice. DEX coadministration, up to a maximum dose of 50 mg/kg/day, did not prevent the induction of these parameters. Acyl-CoA oxidase mRNA levels increased 5-fold with WY14,643 treatment and 15-fold with DEX coadministration at 5 mg/kg/day. ApoCIII mRNA levels were decreased by 50% in WY14,643-treated mice. DEX alone at 5 mg/kg/day increased the ApoCIII mRNA 4-fold, but WY14,643 coadministration also inhibited this induction by greater than 50%. In addition, immunohistochemical detection of peroxisomes with anti-PMP-70 antibody demonstrated marked increase in hepatocellular peroxisomes in WY14,643-treated mice regardless of DEX treatment. In contrast, coadministration of DEX at 2 mg/kg/day partially inhibited the hepatocyte proliferation response (measured by BrdU incorporation or Ki-67 immunohistochemical detection). Moreover, DEX at doses of 5 mg/kg/day or higher completely inhibited the induction of cell proliferation and, at these higher doses, reduced the cell proliferation rate to levels below the vehicle-treated control mice. Our studies clearly demonstrate that the hepatocellular proliferation induced by a peroxisome proliferator can be modulated independently of the other pleiotropic effects usually induced by these agents, suggesting an indirect mechanism of hyperplasia.

Prolonged sublethal exposure to the protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects.

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.

Effects of fumonisin-containing culture material on pulmonary clearance in swine.

OBJECTIVE: To determine the potential effects of feeding tumonisin-containing culture material on the pulmonary clearance of blood-borne particulates and bacteria in swine. ANIMALS: 21 healthy male pigs randomly assigned to control and treated groups. PROCEDURE: Control pigs were fed a standard grower ration while culture material containing fumonisins (20 mg of hydrolyzed fumonisin B1/kg of body weight/d) was added to the feed of treated pigs for 7 days. On day 8, pigs were anesthetized with halothane and catheterized, using a sterile cut-down procedure. 18 hours after recovery from anesthesia, Monastral Blue or Pseudomonas aeruginosa was infused into the right atrium of treated and control pigs for 30 minutes and pulmonary clearance was determined. RESULTS: Pigs that were fed fumonisin-containing culture material had a significantly (P < 0.05) decreased ability to clear Monastral Blue and P aeruginosa. Ultrastructural examination of the lung indicated that uptake of copper pigment by pulmonary intravascular macrophages was decreased in treated pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Fumonisins, even when fed to pigs at sub-lethal concentrations, can inhibit pulmonary intravascular macrophages from removing particulate matter and bacteria from the circulation, thus potentially predisposing swine to infectious disease.

Human tumor necrosis factor receptor (p55) and interleukin 10 gene transfer in the mouse reduces mortality to lethal endotoxemia and also attenuates local inflammatory responses.

Anticytokine therapies have been promulgated in gram-negative sepsis as a means of preventing or neutralizing excessive production of proinflammatory cytokines. However, systemic administration of cytokine inhibitors is an inefficient means of targeting excessive production in individual tissue compartments. In the present study, human gene transfer was used to deliver to organs of the reticuloendothelial system antagonists that either inhibit tumor necrosis factor-alpha (TNF-alpha) synthesis or block its interactions with cellular receptors. Mice were treated intraperitoneally with cationic liposomes containing 200 micrograms of either a pCMV (cytomegalovirus)/p55 expression plasmid that contains the extracellular domain and transmembrane region of the human p55 TNF receptor, or a pcD-SR-alpha/hIL-10 expression plasmid containing the DNA for human interleukin 10. 48 h later, mice were challenged with lipopolysaccharide (LPS) and D-galactosamine. Pretreatment of mice with p55 or IL-10 cDNA-liposome complexes improved survival (p < 0.01) to LPS-D-galactosamine. In additional studies, intratracheal administration of IL-10 DNA-liposome complexes 48 h before an intratracheal LPS challenge reduced pulmonary TNF-alpha levels by 62% and decreased neutrophil infiltration in the lung by 55% as measured by myeloperoxidase activity (both p < 0.05). Gene transfer with cytokine inhibitors is a promising option for the treatment of both the systemic and local sequelae of septic shock.

Differential expression of tumor necrosis factor and interleukin-6 by peritoneal macrophages in vivo and in culture.

To investigate the differences in cytokine regulation in vitro as compared to in vivo, we examined the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peritoneal macrophages in response to lipopolysaccharide (LPS). Mice (CBA/J) were primed with an intraperitoneal injection of complete Freund's adjuvant and after 2 weeks, peritoneal cells were harvested for culture or mice were injected intraperitoneally with LPS for in vivo studies. In ascites fluid, TNF-alpha peaked 1 hour after LPS and returned to baseline levels by 4 hours. In contrast, TNF-alpha in the media reached maximum at 7 hours. Expression of TNF-alpha messenger (m)RNA in vivo was rapid but transient, as levels peaked at 15 minutes and returned to baseline 1 hour after LPS. In contrast, TNF-alpha mRNA in vitro became maximal at 1 hour, but remained elevated to 5 hours after LPS. In vivo, IL-6 in ascites fluid peaked at 2 hours, whereas in vitro, IL-6 continued increasing to 24 hours. In vivo, IL-6 mRNA reached maximum at 30 minutes, but fell below baseline by 1.5 hours after LPS. In contrast, IL-6 mRNA in vitro was sustained at maximal expression between 5 to 9 hours after LPS. These results demonstrate that both TNF-alpha and IL-6 synthesis is more rapid in vivo than in vitro. The rapid kinetics of cytokine expression in vivo must considered when designing strategies to inhibit cytokine action in vivo.

Cytokine binding and clearance properties of proteinase-activated alpha 2-macroglobulins.

Multiple mechanisms are necessary to spatially and temporally restrict and direct the effects of potent mediators of inflammation, immune reactions and tissue repair. Recent studies implicate alpha 2-macroglobulin (alpha 2M) as a protein that regulates the distribution and activity of many cytokines, including transforming growth factors-beta (TGFs-beta), tumor necrosis factor-alpha (TNF-alpha), platelet derived growth factor (PDGF), interleukin-6 (IL-6), nerve growth factor (NGF), fibroblast growth factor (b-FGF), and interleukin-1 beta (IL-1 beta). Some cytokines, including PDGF, NGF, and IL-6 bind preferentially to the native secreted form of alpha 2M, whereas the TGF-beta s, TNF-alpha and IL-1 beta bind preferentially to forms of alpha 2M that have been modified by proteinases such as plasmin. Cytokines bound to native alpha 2M retain much of their biologic activity in various bioassays, whereas cytokines bound to "activated" alpha 2Ms have decreased activity in some cell systems. Because native alpha 2M in circulation can escape into extravascular fluid during tissue injury and inflammation, alpha 2M is a putative cytokine carrier, especially in the presence of heparin or specific cytokine receptors that can displace non-covalently bound cytokines from native alpha 2M. However, proteinase or chemically modified alpha 2Ms become activated for receptor-mediated endocytosis (RME) when they undergo conformational alterations that expose a latent alpha 2M receptor-recognition domain. Circulating activated alpha 2Ms, together with bound cytokines, are rapidly removed by hepatic alpha 2M-receptors (alpha 2M-R) but also bind to other cells expressing alpha 2M-R. This suggests that diseases resulting from an apparent change in the production of one or several different cytokines might represent changes in either the production of alpha 2M "cytokine scavengers" or their alpha 2M-receptor-mediated clearance/targeting mechanisms. The sequence identity between the LDL-receptor related protein and the alpha 2M receptor (115) provides a theoretical basis for interference with cytokine clearance by association of competing lipoprotein ligands with this cytokine clearance pathway. Furthermore, activated alpha 2Ms or augmentation of alpha 2M-receptor-dependent cytokine clearance might be novel strategies for preventing the harmful systemic or local effects of excess cytokines such as TGF-beta s and TNF-alpha in vivo.

Binding of tumor necrosis factor alpha to activated forms of human plasma alpha 2 macroglobulin.

We tested the hypothesis that human plasma alpha 2 macroglobulin (alpha 2M) is a latent binding glycoprotein for human tumor necrosis factor alpha (TNF-alpha). Human recombinant 125I-TNF-alpha was incubated for 2 hours (37 degrees C) with purified native alpha 2M and with alpha 2M that was modified by reaction with methylamine or various proteinases. 125I-TNF-alpha/alpha 2M complexes were detected by nondenaturing polyacrylamide gel electrophoresis after autoradiography or by liquid chromatography on Superose-6. 125I-TNF-alpha bound strongly but noncovalently to alpha 2M-plasmin and alpha 2M-methylamine. There was minimal binding of 125I-TNF-alpha to native alpha 2M, alpha 2M-trypsin, or alpha 2M-thrombin. A 10(6) molar excess of porcine heparin did not reduce the binding of 125I-TNF-alpha to alpha 2M-methylamine or alpha 2M-plasmin. alpha 2M-plasmin or alpha 2M-methylamine added to human plasma or serum preferentially bound 125I-TNF-alpha in the presence of native alpha 2M. 125I-TNF-alpha also bound to 'fast' alpha-macroglobulins in methylamine-reacted human, rat, mouse, swine, equine, and bovine plasma. However, TNF-alpha, preincubated with either alpha 2M-plasmin or alpha 2M-methylamine, remained a potent necrogen for cultured L929 cells. Purified 125I-TNF-alpha/alpha 2M-plasmin complex injected intravenously in CD-1 mice rapidly cleared from the circulation, unless the alpha 2M-receptor pathway was blocked by coinjection of excess alpha 2M-trypsin. These findings demonstrate that alpha 2M is a latent plasmin-activated binding glycoprotein for TNF-alpha and that TNF-alpha/alpha 2M-plasmin complexes can be removed from the circulation by the alpha 2M-receptor pathway. This suggests that alpha 2M may be an important regulator of the activity and distribution of TNF-alpha in vivo.

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.

Counteracting effects of dexamethasone and alpha 2-macroglobulin on inhibition of proliferation of normal and neoplastic rat hepatocytes by transforming growth factors-beta type 1 and type 2.

Primary cultures of hepatocytes isolated from normal F-344 rats or from F-344 rats with hepatocellular carcinomas generated by a 2-step model of chemical carcinogenesis were used to determine if dexamethasone (DEX) or alpha 2-macroglobulin (alpha 2M) modify the ability of transforming growth factors-beta type I (TGF-beta I) and type 2 (TGF-beta 2) to inhibit labelling index of hepatocytes cultured continuously with or without epidermal growth factor (EGF). Both TGF-beta 1 and beta 2 were equivalently potent inhibitors of S-phase DNA synthesis in normal and neoplastic hepatocytes as determined by 3H-thymidine autoradiography. Both DEX (1 to 100 microM) and alpha 2M (50-200 microM) partially counteracted the mito-inhibitory effect of both TGF-betas on the proliferation of normal and surrounding hepatocytes. In contrast, neoplastic hepatocytes cultured with DEX released much less immunoreactive alpha 2M and were less able to overcome the inhibitory effect of TGF-beta than normal or surrounding hepatocytes. Purified bovine alpha 2M partially counteracted the inhibition of TGF-beta 1 or beta 2 of both surrounding and neoplastic hepatocytes. Both DEX and alpha 2M were more effective against the mito-inhibitory activity of TGF-beta 2. Our data suggest that alpha 2M released by DEX-treated normal hepatocytes contributes to the counteraction of the TGF-beta effect by DEX. Our results support the hypothesis that glucocorticoids and growth-factor-binding proteins may have important roles in modulating the effects of TGF-beta on normal hepatocyte proliferation and suggest that under some conditions hepatocellular neoplasms can be more sensitive than normal hepatocytes to inhibition of proliferation by TGF-beta.

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.

Alpha 2-macroglobulin and serum preferentially counteract the mitoinhibitory effect of transforming growth factor-beta 2 in rat hepatocytes.

Transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) are potent, multifunctional modifiers of cellular proliferation and differentiation in many cell types. To evaluate factors which may alter the activity of the TGF-beta s during hepatocyte proliferation, we examined the influences of bovine serum and purified bovine or human alpha 2-macroglobulin (alpha 2M) on the mitoinhibitory effects of the TGF-beta in primary cultures of rat hepatocytes. The inhibitory activity of TGF-beta was evaluated by autoradiographic labeling index at 48 hours in hepatocyte cultures exposed to [3H]thymidine between hours 24 and 48 in culture. In the absence of serum or alpha 2M, TGF-beta 1 and TGF-beta 2 were equivalently potent in inhibiting S-phase DNA synthesis in hepatocytes cultured with or without epidermal growth factor. However, bovine serum and purified bovine or human alpha 2M consistently counteracted the mitoinhibitory effects of TGF-beta 2. S-phase DNA synthesis increased five- to six-fold when bovine serum (15%) or alpha 2M (200 micrograms/ml) were included with TGF-beta 2 (0.1 ng/ml) and epidermal growth factor (20 ng/ml). The mitoinhibitory effect of TGF-beta 1 was not influenced by the addition of purified bovine alpha 2M. Human alpha 2M or bovine serum counteracted inhibition by TGF-beta 1 to a lesser extent. [125I]TGF-beta 1 and 125I]TGF-beta 2 formed complexes with purified bovine alpha 2M and serum proteins migrating at identical positions to purified alpha 2M during nondenaturing polyacrylamide gel electrophoresis. However, [125I]TGF-beta 1 associated preferentially with the "fast" migrating form of alpha 2M, whereas [125I]TGF-beta 2 associated with both the "slow" and "fast" migrating forms. Inhibitory activity of TGF-beta 1 and TGF-beta 2 coeluted with alpha 2M in the high molecular weight fractions from a Sephacryl S-200 column. These results support the hypothesis that purified alpha 2M and alpha 2M in bovine serum binds both TGF-betas but preferentially counteracts the mitoinhibitory effect of TGF-beta 2 on rat hepatocytes.

Reversible mitochondrial swelling in cultured rat hepatocytes exposed to 1,2-dimethylhydrazine.

The early structural changes of F344 rat hepatocytes exposed to the hepatocarcinogen 1,2-dimethylhydrazine (DMH) were characterized in short-term monolayer cultures. Continuous exposure of monolayers to DMH (2-16 mM) caused cytoplasmic vacuoles visible by phase-contrast microscopy in all hepatocytes within 6 hr of exposure. These changes preceded maximal release of lactate dehydrogenase (LDH) which occurred after 48 hr of continuous exposure to cytocidal concentrations of DMH (8-16 mM). Ultrastructurally, hepatocytes exposed to DMH (4 mM, 6 hr) showed a twofold increase in mitochondrial diameter from 340 +/- 70 nm in control hepatocytes to 800 +/- 140 nm in DMH-exposed cells. Hepatocyte monolayers exposed to DMH (4 mM, 6 hr) with subsequent removal of DMH attained normal phase-contrast appearance within 6 hr. Ultrastructural studies showed no significant differences when compared with control hepatocytes and mitochondrial diameters (330 +/- 70 nm) were comparable with control hepatocytes. Pretreatment of hepatocytes with depletors of cellular reduced glutathione concentration, including 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM) and diethyl maleate (160 microM), did not potentiate hepatocellular vacuolation nor release of LDH from hepatocytes exposed to DMH (0-16 mM, 48 hr). These studies demonstrate a distinctive form of reversible high-amplitude mitochondrial swelling that can be monitored by phase-contrast microscopy of cultured hepatocytes in monolayers. Since DMH-induced mitochondrial swelling and its progression to irreversible injury are not potentiated by depletors of reduced thiols, this response appears distinct from prelethal mitochondrial swelling in hepatocytes subjected to oxyradical-mediated mechanisms of injury.

Inverse relationship between epidermal growth factor induced proliferation and expression of high affinity surface epidermal growth factor receptors in rat hepatocytes.

Rat hepatocytes express large numbers of high and low affinity surface membrane receptors (EGFR) for epidermal growth factor (EGF) but the roles of EGF and EGFRs in hepatocyte proliferation in vivo are unclear. F344 rat hepatocytes in primary culture proliferated maximally in response to continuous serum-free culture with 3.3 nM (20 ng/ml) EGF, as quantified by cumulative [3H]thymidine labeling index. However, serum concentrations of EGF in rats with normal livers or induced hepatocyte proliferation due to partial hepatectomy, carbon tetrachloride-induced necrosis, or hepatic neoplasia were consistently below 0.1 nM. The 3- or 6-hour pulse exposures to EGF (1.7 nM) between 0 to 16 hours had minimal effect on labeling index at 48 hours, but these pulse exposures at 24 or 32 hours were equivalent to continuous exposure. At 24 and 32 hours, the total specific surface binding of [125I]EGF to hepatocytes cultured free of EGF decreased to 43 and 24% of the initial values, respectively. Scatchard analysis of EGF binding indicated that hepatocytes lost all high affinity EGFRs (Kd of 0.08 nM) by 24 hours. Low affinity [125I]EGF binding at 0 hour (Kd 0.8 nM) was further reduced at 24 hours (Kd = 3.9 nM) and corresponded more closely to mitogenic concentrations of EGF in culture. These studies demonstrate that proliferative responsiveness of hepatocytes to EGF increases during culture by a process that involves prior loss of constitutive high affinity EGFRs. These results suggest that constitutive high affinity EGFRs do not elicit the proliferative response to EGF.

Inhibition of proliferation of normal, preneoplastic, and neoplastic rat hepatocytes by transforming growth factor-beta.

The influences of purified transforming growth factor beta (TGF-beta) on proliferation of normal, preneoplastic, and neoplastic rat hepatocytes were examined in primary monolayer culture with or without prior stimulation with epidermal growth factor (EGF). Hepatocytes from normal livers or discrete preneoplastic nodules or carcinomas generated in F344 rats by the Solt-Farber model were isolated and cultured in serum-free modified Williams' E. medium for up to 72 h. Proliferation was quantified by labeling index by [3H]thymidine autoradiography. The majority of normal hepatocytes became labeled in response to EGF (20 ng/ml) between 24 and 72 h. TGF-beta had a dose-dependent inhibitory effect which was virtually complete at concentrations above 0.5 ng/ml added at 0 h together with EGF. Hepatocytes from all nodule and carcinoma populations were less stimulated by EGF but also strongly inhibited by TGF-beta. Hepatocytes isolated from normal livers 24 h after partial hepatectomy were similarly inhibited by TGF-beta. The minimal initial exposure period for TGF-beta to maximally inhibit was 2 h. TGF-beta added at various times between 8 and 48 h after EGF partially inhibited the labeling index to levels that were constant but substantially greater than the labeling index at the time TGF-beta was added. A proportion of hepatocytes from normal and nodular livers became resistant to the inhibitory effects of TGF-beta between 48 and 72 h, suggesting that the inhibitory effect is transient. TGF-beta added at 0 h also virtually completely inhibited the labeling of normal and nodular hepatocytes that were not exposed to EGF. These studies demonstrate that TGF-beta is a potent negative regulator of proliferation of normal, regenerating, preneoplastic, and neoplastic hepatocytes. This suggests that persistent proliferation of neoplastic hepatocytes in vivo cannot be explained by a difference in response to TGF-beta.