Doxycycline alters collagen composition following ventral hernia repair.

BACKGROUND: Doxycycline, a nonspecific metalloproteinase (MMP) inhibitor, has been demonstrated to impact the strength of the polypropylene (PP) mesh-repaired hernia with an increase in the deposition of collagen type 1. The impact of doxycycline with porcine acellular dermal matrices (PADM) is unknown; therefore, we evaluated the impact of doxycycline administration upon hernia repair with PP and PADM mesh. METHODS: Sprague-Dawley rats weighing ~400 g underwent laparotomy with creation of a midline ventral hernia. After a 27-day recovery, animals were randomly assigned to four groups of eight and underwent intraperitoneal underlay hernia repair with either PP or PADM. Groups were assigned to daily normal saline (S) or daily doxycycline in normal saline 10 mg/kg (D) via oral gavage for 8 weeks beginning 24 h preoperatively. Animals were euthanized at 8 weeks and underwent tensiometric testing of the abdominal wall and western blot analyses for collagen subtypes and MMPs. RESULTS: Thirty-two animals underwent successful hernia creation and repair with either PADM or PP. At 8 weeks, 15 of 16 PP-implanted animals survived with only 12 of 16 PADM-implanted animals surviving. There were no differences in the mesh to fascial interface tensiometric strength between groups. Densitometric counts in the PADM-D group demonstrated increased collagen type 1 compared to PP-S (PADM-D [1286.5], PADM-S [906.9], PP-S [700.4], p = 0.037) and decreased collagen type 3 compared to PP-S (PADM-D [7446.9], PADM-S [8507.6], PP-S [11,297.1], p = 0.01). MMP-9 levels were increased in PADM-D (PP-S vs. PADM-D, p = 0.04), while MMP-2 levels were similar between PADM-D and PADM-S, respectively. CONCLUSIONS: Collagen type 1 deposition at the mesh to fascial interface is enhanced following administration of doxycycline in ventral hernia repairs with porcine acellular dermal matrices. Doxycycline administration may have implications for enhancing hernia repair outcomes using biologic mesh.

Doxycycline shows dose-dependent changes in hernia repair strength after mesh repair.

BACKGROUND: Ventral hernia is a commonly occurring surgical problem. Our earlier studies have shown that a 30 mg/kg dose of doxycycline can significantly impact the strength of polypropylene (PP) mesh in a rat hernia repair model at 6 and 12 weeks. The objective of the present study was to investigate the dose dependence of doxycycline treatment on hernia repair strengths in rats. STUDY DESIGN: Fifty-six Sprague-Dawley rats underwent hernia repair with either PP mesh (n = 28) or sutures only (primary; n = 28); both groups were further divided into four doxycycline groups of seven animals each: control (0 mg/kg), low (3 mg/kg), medium (10 mg/kg), and high (30 mg/kg). One day before hernia repair surgery, animals received doxycycline doses by gavage and continued receiving daily until euthanasia. After 8 weeks, rats were euthanized and tissue samples from hernia repaired area were collected and analyzed for tensile strength using a tensiometer (Instron, Canton, MA, USA), while MMPs 2, 3, and 9, and collagen type 1 and 3 were analyzed by western blotting. RESULTS: In mesh-repaired animals, medium and high doxycycline dose repaired mesh fascia interface (MFI) showed significant increase in tensile strength when compared to control. In the primary repaired animals, there was no significant difference in MFI tensile strength in any dose group. In medium-dose MFI, there was a significant reduction in MMPs 2, 3, and 9. In this animal group, MFI showed significant increase in collagen 1 and significant reduction in collagen type 3 when compared to control. CONCLUSION: It is possible to improve the strength of mesh-repaired tissue by administering a significantly lower dose of the drug, which has implications for translation of the findings.

Doxycycline administration improves fascial interface in hernia repair.

BACKGROUND: Despite improvements in ventral hernia repair techniques, their recurrence rates are unacceptably high. Increased levels of matrix metalloproteinases (MMPs) and reduced collagen-1 to -3 ratios are implicated in incisional hernia formation. We have recently shown doxycycline treatment for 4 wk after hernia repair reduced MMP levels, significantly increased collagen-1 to -3 ratios, and increased tensile strength of repaired interface fascia. However, this increase was not statistically significant. In this study, we extended treatment duration to determine whether this would impact the tensile strength of the repaired interface fascia. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats underwent incision hernia creation and subsequent repair with polypropylene mesh. The animals received either saline (n = 16) or doxycycline (n = 16) beginning from 1 day before hernia repair until the end of survival time of 6 wk (n = 16) or 12 wk (n = 16). Tissue samples were investigated for MMPs and collagen subtypes using Western blot procedures, and tensiometric analysis was performed. RESULTS: At both 6 and 12 wk after hernia repair, the tensiometric strength of doxycycline-treated mesh to fascia interface (MFI) tissue showed a statistically significant increase when compared with untreated control MFI. In both groups, collagen-1, -2, and -3 ratios were remarkably increased in doxycycline-treated MFI. At 6 wk, the doxycycline-treated MFI group showed a significant decrease in MMP-2, an increase in MMP-3, and no change in MMP-9. At 12 wk, MMP-9 showed a remarkable reduction, whereas MMP-2 and -3 protein levels increased in the doxycycline-treated MFI group. CONCLUSIONS: Doxycycline administration results in significantly improved strength of repaired fascial interface tissue along with a remarkable increase in collagen-1, -2, and -3 ratios.

Doxycycline impacts hernia repair outcomes.

BACKGROUND: Incisional hernias occur commonly in up to 20% of all abdominal operations. Incisional hernia formation has been associated with increased levels of matrix metalloproteinases (MMPs), reduced collagen 1, and increased collagen 3 expression. Doxycycline, a nonspecific inhibitor of MMPs, has been shown to beneficially reduce MMP levels in both cancer and aneurysm models. This study evaluates the impact of doxycycline upon MMP expression, collagen subtypes, and hernia repair distraction forces in an animal model of incisional hernia repair. MATERIALS AND METHODS: Twenty-four Sprague Dawley rats underwent incisional hernia creation and subsequent repair with polypropylene mesh. Animals were administered doxycycline or saline daily beginning 1 d prior to hernia repair and survived for 1, 2, or 4 wk. Serum and tissue were evaluated for MMP content and collagen subtyping utilizing enzyme-linked immunosorbent assay and Western blot. Tensiometric properties of the native abdominal wall after hernia repair were measured with an Instron Corp. (Canton, MA) mechanical testing system. RESULTS: There were no differences in control and experimental groups 1 and 2 wk following hernia repair; 4 wk following hernia repair, doxycycline treated animals demonstrated reduced serum MMP-2 and MMP-9 levels, reduced tissue levels of MMP-2, MMP-3, and MMP-9, and increased collagen 1 to 3 ratios. Distraction forces required to disrupt the hernia repair were increased in the doxycycline treated group compared with controls. CONCLUSIONS: Doxycycline administration is associated with improved hernia repair strength with concomitant reduction of MMP levels with increased collagen 1 deposition. Longer term studies are required to better understand the impact of this treatment.

Effects of cigarette smoke on the activation of oxidative stress-related transcription factors in female A/J mouse lung.

Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine whether cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 d (6 hr/d, 5 d/wk). Cigarette smoke did not increase NF-kappaB activation at any of these times, but NF-kappaB DNA binding activity was lower after 15 d and 56 d of smoke exposure. The DNA binding activity of AP-1 was lower after 10 d and 56 d but was not changed after 42 d of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 d of smoke exposure but decreased after 56 d. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 d of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-kappaB, AP-1, and HIF are largely unaffected or reduced.

Subacute exposure to N-ethyl perfluorooctanesulfonamidoethanol results in the formation of perfluorooctanesulfonate and alters superoxide dismutase activity in female rats.

Perfluorooctanesulfonamides, such as N-ethyl perfluorooctanesulfonamidoethanol (N-EtFOSE), are large scale industrial chemicals but their disposition and toxicity are poorly understood despite significant human exposure. The hypothesis that subacute exposure to N-EtFOSE, a weak peroxisome proliferator, causes a redox imbalance in vivo was tested using the known peroxisome proliferator, ciprofibrate, as a positive control. Female Sprague-Dawley rats were treated orally with N-EtFOSE, ciprofibrate or corn oil (vehicle) for 21 days, and levels of N-EtFOSE and its metabolites as well as markers of peroxisome proliferation and oxidative stress were assessed in serum, liver and/or uterus. The N-EtFOSE metabolite profile in liver and serum was in good agreement with reported in vitro biotransformation pathways in rats and the metabolite levels decreasing in the order perfluorooctanesulfonate >> perfluorooctanesulfonamide ~ N-ethyl perfluorooctanesulfonamidoacetate >> perfluorooctanesulfonamidoethanol approximately N-EtFOSE. Although N-EtFOSE treatment significantly decreased the growth rate, increased relative liver weight and activity of superoxide dismutases (SOD) in liver and uterus (total SOD, CuZnSOD and MnSOD), a metabolic study revealed no differences in the metabolome in serum from N-EtFOSE-treated and control animals. Ciprofibrate treatment increased liver weight and peroxisomal acyl Co-A oxidase activity in the liver and altered antioxidant enzyme activities in the uterus and liver. According to NMR metabolomic studies, ciprofibrate treated animals had altered serum lipid profiles compared to N-EtFOSE-treated and control animals, whereas putative markers of peroxisome proliferation in serum were not affected. Overall, this study demonstrates the biotransformation of N-EtFOSE to PFOS in rats that is accompanied by N-EtFOSE-induced alterations in antioxidant enzyme activity.

Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-kappa B in mice.

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-kappaB on the hepatic tumor promoting activity of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50-/- male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 micromol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P=0.09) increased the tumor incidence in wild-type and p50-/- mice. PCB-153 increased the total tumor volume in both wild-type and p50-/- mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50-/- mice administered PCB-153 compared to vehicle controls, and inhibited in p50-/- mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50-/- mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50-/- mice; this increase was inhibited in p50-/- mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P=0.10) increased apoptosis in p50-/- but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-kappaB p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.

Effect of dietary selenium on the promotion of hepatocarcinogenesis by 3,3', 4,4'-tetrachlorobiphenyl and 2,2', 4,4', 5,5'-hexachlorobiphenyl.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have promoting activity in the liver. PCBs induce oxidative stress, which may influence carcinogenesis. Epidemiological studies strongly suggest an inverse relationship between dietary selenium (Se) and cancer. Despite evidence linking Se deficiency to hepatocellular carcinoma and liver necrosis, the underlying mechanisms for Se cancer protection in the liver remain to be determined. We examined the effect of dietary Se on the tumor promoting activities of two PCBs congeners, 3,3', 4,4'-tetrachlorobiphenyl (PCB-77) and 2,2', 4,4', 5,5'-hexachlorobiphenyl (PCB-153) using a 2-stage carcinogenesis model. An AIN-93 torula yeast-based purified diet containing 0.02 (deficient), 0.2 (adequate), or 2.0 mg (supplemental) selenium/kg diet was fed to Sprague-Dawley female rats starting ten days after administering a single dose of diethylnitrosamine (150 mg/kg). After being fed the selenium diets for 3 weeks, rats received four i.p. injections of either PCB-77 or PCB-153 (150 micromol/kg) administered every 14 days. The number of placental glutathione S-transferase (PGST)-positive foci per cm(3) and per liver among the PCB-77-treated rats was increased as the Se dietary level increased. Unlike PCB-77, rats receiving PCB-153 did not show the same Se dose-response effect; nevertheless, Se supplementation did not confer protection against foci development. However, the 2.0 ppm Se diet reduced the mean focal volume, indicating a possible protective effect by inhibiting progression of preneoplastic lesions into larger foci. Cell proliferation was not inhibited by Se in the liver of the PCB-treated groups. Se did not prevent the PCB-77-induced decrease of hepatic Se and associated reduction in glutathione peroxidase (GPx) activity. In contrast, thioredoxin reductase (TrxR) activity was not affected by the PCBs treatment or by Se supplementation. These findings indicate that Se does not inhibit the number of PGST-positive foci induced during promotion by PCBs, but that the size of the lesions may be inhibited. The effects of Se on altered hepatic foci do not correlate with its effects on GPx and TrxR.

Effect of antioxidant phytochemicals on the hepatic tumor promoting activity of 3,3',4,4'-tetrachlorobiphenyl (PCB-77).

Polychlorinated biphenyls (PCBs) have promoting activity in the liver, which may be brought about in part by the induction of oxidative stress. In this study we examined the effects of several antioxidant phytochemicals on the tumor promoting activity of 3,3',4'4-tetrachlorobiphenyl (PCB-77). Female Sprague Dawley rats were first injected with diethylnitrosamine (DEN, 150 mg/kg) and one week later the rats were fed an AIN-93 based purified diet or the same diet containing ellagic acid (0.4%), beta-carotene (0.5%), curcumin (0.5%), N-acetyl cysteine (NAC, 1.0%), coenzyme CoQ10 (CoQ10, 0.4%), resveratrol (0.005%), lycopene (10% as Lycovit, which contains 10% lycopene), or a tea extract (1%, containing 16.5% epigallocatechin-3-gallate [EGCG] and 33.4% total catechins). Rats were fed the diets for the remainder of the study. After three weeks, 2/3 of the control rats and all of the antioxidant diet-fed rats were injected i.p. with PCB-77 (300 micromol/kg) every other week for four injections. All rats were euthanized ten days after the last PCB injection. The rats that received PCB-77 alone showed an increase in the number and size of placental glutathione S-transferase (PGST)-positive foci in the liver. Lycopene significantly decreased the number of foci, while curcumin and CoQ10 decreased the size of the foci. In contrast, ellagic acid increased the number but decreased the size of the foci. All of the other phytochemicals showed only slight or no effects. Compared with the PCB-77 group, CoQ10 increased cell proliferation in normal hepatocytes, whereas the other antioxidants had no effect in either normal or PGST-positive hepatocytes. These findings show that none of the antioxidant phytochemicals produced a clear decrease in the promoting activity of PCB-77.

Role of oxidative stress in the promoting activities of pcbs.

PCBs are organic pollutants that persist and bioaccumulate in the environment. These chemicals induce and promote liver tumors in rodents. Previous studies have shown that they increase oxidative stress in the liver, including lipid peroxidation, oxidative DNA damage, and NF-κB activation. The objective of these studies was to determine if the promoting activities of PCBs could be inhibited by dietary antioxidants (vitamin E, selenium, or phytochemicals) or by knocking out the p50 subunit of NF-κB. In the antioxidant studies, female rats were first injected with DEN (150 mg/kg) and then administered 4 biweekly i.p. injections (300 µmol/kg/injection) of PCB-77, PCB-153, or vehicle; the number and volume of placental glutathione S-transferase (PGST)-positive foci were then quantified. Vitamin E did not influence the promoting activities of PCBs. Increasing dietary selenium above the recommended intake increased the number of foci induced but decreased their volume. Most of the phytochemicals examined (N-acetyl cysteine, ß-carotene, resveratrol, EGCG) had no significant effect on the promoting activity of PCB-77. Ellagic acid increased and lycopene decreased the number of foci; ellagic acid, CoQ(10), and curcumin decreased the volume of foci. In the NF-κB knockout study, male mice were first injected with DEN (90 mg/kg); controls not receiving DEN were also studied. Both p50 -/- and wild-type mice were then injected biweekly 20 times with PCB-153 (300 (µmol/kg). In DEN-treated and DEN + PCB-treated mice, the incidence of tumors was lower in the p50 -/- mice than in wild-type mice. In mice receiving PCB-153, the tumor incidence and tumor volume were higher. The volume of tumors that were positive for glutamine synthetase was increased in mice administered PCB-153. This study shows that the promotion of hepatocarcinogenesis by PCBs is largely unaffected by dietary antioxidants but is diminished when NF-κB activation is impaired by the absence of the p50 subunit.

The effect of dietary glycine on the hepatic tumor promoting activity of polychlorinated biphenyls (PCBs) in rats.

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3',4,4'-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague-Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 micromol/kg), PCB-153 (300 micromol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153.

Inhibition of hepatocarcinogenesis by the deletion of the p50 subunit of NF-kappaB in mice administered the peroxisome proliferator Wy-14,643.

Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic peroxisome proliferation and tumors in rodents. We previously showed that peroxisome proliferators increase NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines, and that mice deficient in the p50 subunit of NF-kappaB had much lower cell proliferation in response to the peroxisome proliferator ciprofibrate. In this study we examined the promotion of hepatocarcinogenesis by WY in the p50 knockout (-/-) mice. The p50 -/- and wild type mice were first administered diethylnitrosamine (DEN) as an initiating agent. Mice were then fed a control diet or a diet containing 0.05% WY for 38 weeks. Wild-type mice receiving DEN only developed a low incidence of tumors, and the majority of wild-type mice receiving both DEN and WY developed tumors. However, no tumors were seen in any of the p50 -/- mice. Cell proliferation and apoptosis were measured in hepatocytes by BrdU labeling and the TUNEL assay, respectively. Treatment with DEN + WY increased both cell proliferation and apoptosis in both the wild-type and p50 -/- mice; DEN treatment alone has no effect. In the DEN/WY-treated mice, cell proliferation and apoptosis were slightly lower in the p50 -/- mice than in the wild-type mice. These data demonstrate that NF-kappaB is involved in the promotion of hepatic tumors by the peroxisome proliferator WY; however, the difference in tumor incidence could not be attributed to alterations in either cell proliferation or apoptosis.

PCB 153, a non-dioxin-like tumor promoter, selects for beta-catenin (Catnb)-mutated mouse liver tumors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental toxicants which act as liver tumor promoters in rodents and can be classified as either dioxin-like or non-dioxin (phenobarbital [PB])-like inducers of cytochrome P-450. Since we have previously shown that tumor promotion by PB leads to clonal outgrowth of beta-catenin (Catnb)-mutated but not Ha-ras-mutated mouse liver tumors, we were interested to know whether the non-dioxin-like tumor promoter 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) shows the same selective pressure during tumor promotion. Male B6129SF2/J mice were given a single injection of N-nitrosodiethylamine (90 mg/kg body weight) at 9 weeks of age, followed by 39 weeks of treatment with PCB 153 (20 biweekly ip injections of 300 mumol/kg body weight) or corn oil as a control. Animals were killed 15 weeks after the last PCB 153 injection and liver tumors were identified by immunohistochemical staining of glutamine synthetase (GS) and analyzed for Catnb, Ha-ras, and B-raf mutations. Quantitative analyses revealed that GS-positive tumors were much larger and more frequent in livers from PCB 153-treated mice than in control animals, whereas GS-negative tumors were similar in both groups. Almost 90% (34/38) of all tumors from PCB 153-treated animals contained Catnb mutations, which compares to approximately 45% (17/37) of tumors in the control group. Ha-ras- and B-raf-mutated liver tumors were rare and not significantly different between treatment groups. These results clearly indicate that PCB 153 strongly selects for Catnb-mutated, GS-positive liver tumors, which is similar to the known action of PB, a prototypical tumor promoter in rodent liver.

Cell proliferation and apoptosis are altered in mice deficient in the NF-kappaB p50 subunit after treatment with the peroxisome proliferator ciprofibrate.

We previously showed that the peroxisome proliferator ciprofibrate increases hepatic NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines. Here, we analyzed the response to ciprofibrate in mice that lack the NF-kappaB p50 gene (p50-/-). Wild-type and p50-/- mice were fed a diet with or without 0.01% ciprofibrate for 10 days. NF-kappaB DNA binding activity was present and increased after ciprofibrate treatment in wild-type mice, but was not detected in p50-/- mice. The untreated p50-/- mice had a higher level of hepatic cell proliferation, as measured by BrdU labeling, than did untreated wild-type mice. However, the increase in proliferation was greater in ciprofibrate-fed wild-type mice than in ciprofibrate-fed p50-/- mice. The apoptotic index was low in wild-type mice in the presence or absence of ciprofibrate. Apoptosis was increased in untreated p50-/- mice compared to wild-type mice; apoptosis was reduced in p50-/- mice after ciprofibrate feeding. The c-Jun and JunB mRNA levels were higher in untreated p50-/- mice than in untreated control mice; c-Jun mRNA levels increased, whereas JunB mRNA levels decreased in both groups after ciprofibrate treatment. The c-Jun and JunB protein levels were the same in untreated wild-type and p50-/- mice and increased in both groups after ciprofibrate treatment. Several apoptosis-related mRNAs were higher in untreated p50-/- mice compared to untreated control mice; expression of these genes increased in both groups after ciprofibrate treatment. These data indicate that NF-kappaB contributes to the proliferative and apoptotic changes that occur in the liver in response to ciprofibrate.

Dietary selenium fails to influence cigarette smoke-induced lung tumorigenesis in A/J mice.

The goal of the study was to determine if dietary selenium inhibited the induction of lung tumorigenesis by cigarette smoke in A/J mice. Purified diets containing 0.15, 0.5, or 2.0mg/kg selenium in the form of sodium selenite were fed to female A/J mice. Half of the mice in each dietary group were exposed to cigarette smoke 6h/day, 5days/week for five months followed by a four month recovery period in ambient air, while the other half were used as controls. After the recovery period, the mice were euthanized, and their lungs were removed for further analysis. Mice exposed to smoke had a higher tumor incidence and a higher tumor multiplicity, whereas dietary Se did not affect either the tumor incidence or tumor multiplicity. An increase in dietary selenium led to increased levels of selenium in the lung as well as GPx protein levels, but dietary Se did not affect lung SOD protein levels. In conclusion, these data confirm the carcinogenic activity of cigarette smoke in mice but show that dietary Se provided as sodium selenite does not affect smoke-induced carcinogenesis in this model.

Effect of vitamin E on hepatic cell proliferation and apoptosis in mice deficient in the p50 subunit of NF-κB after treatment with phenobarbital.

Phenobarbital (PB) is an efficacious and well-studied hepatic tumor promoting agent. Nuclear factor-κB (NF-κB) is a transcription factor activated by reactive oxygen and is involved in cell proliferation and apoptosis. We previously found that PB activates NF-κB and that dietary vitamin E is effective in decreasing PB-induced NF-κB DNA binding. We therefore hypothesized that dietary vitamin E influences PB-induced changes in cell proliferation and apoptosis through its action on NF-κB. NF-κB1 deficient mice (p50-/-) and wild-type B6129 mice were fed a purified diet containing 10 or 250ppm vitamin E (α-tocopherol acetate) for 28days. At that time, half of the wild-type and half of the p50-/- mice were placed on the same diet with 0.05% PB for 10days. Compared to wild-type mice, the p50-/- mice had higher levels of cell proliferation and apoptosis. Cell proliferation was significantly increased by PB, but vitamin E did not affect hepatic cell proliferation. Apoptosis was not changed in mice fed PB, and there was no significant difference in apoptosis between control and high vitamin E treated mice. Thus, vitamin E does not appear to influence cell growth parameters in either wild-type or p50-/- mice.

Role of oil vehicle on hepatic cell proliferation in PCB-treated rats.

We report the role of dietary glycine and the type of oil used as a vehicle in the hepatotoxicity of control rats and rats treated with 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153). In our first experiment, glycine or valine (as control) was fed in an unrefined diet at 5% for the entire study duration (5 days) to inhibit Kupffer cell activity. PCB-153 (100 or 300 µmol/kg) dissolved in medium chain triglyceride (MCT) oil, was injected intraperitoneally 2 days before euthanasia; the peroxisome proliferator Wy-14,643 was included as a positive control. MCT oil decreased cell proliferation by approximately 50%. PCB-153 slightly increased hepatic cell proliferation, but dietary glycine did not reduce cell proliferation. Because of the inhibition of cell proliferation in rats receiving MCT oil compared with rats receiving no injection, we hypothesized that MCT oil may have been inhibiting the hepatocyte proliferation in PCB-153-treated rats. We therefore performed another experiment using 3 types of oil as a vehicle for PCB-153: MCT oil, corn oil, and olive oil. Rats were injected with PCB-153 (300 µmol/kg) or one of the vehicles, again 2 days before euthanasia. MCT oil again decreased the hepatocyte proliferation by approximately 50%. In rats receiving PCB-153, hepatocyte proliferation was slightly higher than their respective vehicle controls for corn oil and olive oil but not for MCT oil. These studies show that the oil vehicle is important in cell proliferation after PCB exposure, with MCT oil appearing to be protective.

Dietary antioxidants in the prevention of hepatocarcinogenesis: a review.

In this review, the role of dietary antioxidants in the prevention of hepatocarcinogenesis is examined. Both human and animal models are discussed. Vitamin C, vitamin E, and selenium are antioxidants that are essential in the human diet. A number of non-essential chemicals also contain antioxidant activity and are consumed in the human diet, mainly as plants or as supplements, including beta-carotene, ellagic acid, curcumin, lycopene, coenzyme Q(10), epigallocatechin gallate, N-acetyl cysteine, and resveratrol. Although some human and animal studies show protection against carcinogenesis with the consumption of higher amounts of antioxidants, many studies show no effect or an enhancement of carcinogenesis. Because of the conflicting results from these studies, it is difficult to make dietary recommendations as to whether consuming higher amounts of specific antioxidants will decrease the risk of developing hepatocellular carcinoma.

Effect of dietary selenium and cigarette smoke on pulmonary cell proliferation in mice.

The objective of this study was to determine if dietary selenium could inhibit pulmonary cell proliferation in control and cigarette smoke-exposed female A/J mice. Selenium in the form of sodium selenite was supplemented to purified diets similar to the AIN-93M diet to yield 0.15, 0.5, or 2.0 mg selenium/kg diet. After 3 weeks, mice in each dietary group were divided into two subgroups; one used as control, whereas the other was exposed to cigarette smoke for five consecutive days. Mice from both groups were euthanized 3 days later. Mice were administered bromodeoxyuridine in the drinking water starting 5 days before the initiation of the smoke exposure and continuing until they were euthanized. After euthanasia, the left lung lobe was processed for histology and cell proliferation analysis. Cigarette smoke increased cell proliferation in the terminal bronchioles and large airways, but not in alveoli. High-selenium diets inhibited cell proliferation in the alveoli, terminal bronchioles and large airways areas in both control and smoke-exposed mice. Increasing the dietary selenium level led to increased selenium levels in the blood and lung, and increased glutathione peroxidase (GPx) activity in the lung. Cytochrome P-450 1A1 protein levels in the lung were increased by cigarette smoke but were not affected by dietary selenium. It is concluded that dietary selenium inhibits pulmonary cell proliferation in both control and cigarette smoke-exposed mice, indicating that selenium is inhibiting cell proliferation independently of smoke exposure, and that this inhibition may be related to selenium concentration and GPx activity in the lung.

Effect of phenobarbital on hepatic cell proliferation and apoptosis in mice deficient in the p50 subunit of NF-kappaB.

Phenobarbital (PB) is a nongenotoxic tumor promoter in the liver. One mechanism by which PB may exert its tumor promoting activity is by inducing oxidative stress. We previously found that PB administration increased hepatic NF-kappaB DNA binding activity. In this study we examined the hypothesis that the effects of PB on cell proliferation and apoptosis are dependent on NF-kappaB. We used a mouse model that is deficient in the p50 subunit of NF-kappaB; previous studies had found that p50-/- mice were less sensitive to the induction of hepatic cell proliferation by PCBs or peroxisome proliferators. Mice (p50-/- and wild-type B6129) were fed a control diet or one containing 0.05% PB for 3, 10 or 34 days. At the end of the experiment, the mice were euthanized and livers removed and processed. PB increased cell proliferation at 3 and 10 days (but not at 34 days), but the deletion of the NF-kappaB p50 subunit did not inhibit these increases. p50-/- Mice had higher cell proliferation at the 3 day (only in mice fed PB) and 34-day timepoints. PB decreased hepatocyte apoptosis after 3 days, slightly decreased it after 10 days, and did not affect it after 34 days. The deletion of the NF-kappaB p50 subunit did not influence PB's effect on apoptosis. In p50-/- mice, apoptosis was increased after 3 or 10 days compared to wild-type mice, but no effect was seen after 34 days. The hepatic expression of the NF-kappaB-regulated gene TNF-alpha correlated more with the hepatic cell proliferation data than with hepatic apoptosis, and was not decreased by the deletion of the p50 subunit. These findings show that the p50 subunit of NF-kappaB is not required for the alteration of hepatocyte proliferation or apoptosis by PB up to 34 days after its administration.