Mesenchymal stem cells to treat diabetic neuropathy: a long and strenuous way from bench to the clinic.

As one of the most common complications of diabetes, diabetic neuropathy often causes foot ulcers and even limb amputations. Inspite of continuous development in antidiabetic drugs, there is still no efficient therapy to cure diabetic neuropathy. Diabetic neuropathy shows declined vascularity in peripheral nerves and lack of angiogenic and neurotrophic factors. Mesenchymal stem cells (MSCs) have been indicated as a novel emerging regenerative therapy for diabetic neuropathy because of their multipotency. We will briefly review the pathogenesis of diabetic neuropathy, characteristic of MSCs, effects of MSC therapies for diabetic neuropathy and its related mechanisms. In order to treat diabetic neuropathy, neurotrophic or angiogenic factors in the form of protein or gene therapy are delivered to diabetic neuropathy, but therapeutic efficiencies are very modest if not ineffective. MSC treatment reverses manifestations of diabetic neuropathy. MSCs have an important role to repair tissue and to lower blood glucose level. MSCs even paracrinely secrete neurotrophic factors, angiogenic factors, cytokines, and immunomodulatory substances to ameliorate diabetic neuropathy. There are still several challenges in the clinical translation of MSC therapy, such as safety, optimal dose of administration, optimal mode of cell delivery, issues of MSC heterogeneity, clinically meaningful engraftment, autologous or allogeneic approach, challenges with cell manufacture, and further mechanisms.

Immature dendritic cells expressing indoleamine 2,3-dioxygenase suppress ovalbumin-induced allergic airway inflammation in mice.

BACKGROUND: Proliferation of activated CD4+ T lymphocytes is inhibited by indoleamine 2,3-dioxygenase (IDO). OBJECTIVE: We undertook the present study to test the hypothesis that IDO-expressing immature DCs (imDCs) can restore immune tolerance in mice suffering from allergic airway inflammation. METHODS: imDCs were generated from murine bone marrow cells using granulocyte-macrophage colony-stimulating factor.The imDCs were subsequently transfected with an IDO expression vector (pEGFP-N1-IDO). Surface marker expression, including CD11c, MHCII, CD80, and CD86, was analyzed using flow cytometry. IDO-expressing imDCs were injected into the trachea of ovalbumin (OVA)-sensitized mice, and lung histopathology and cytokine expression in bronchoalveolar lavage fluid were assessed. The splenic CD4+ T cells of OVA-sensitized mice were isolated and co-cultured with pEGFP-N1-IDO-expressing imDCs, and apoptosis of CD4+ T cells was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Expression of IDO in imDCs did not alter cell surface molecule expression. We observed marked lung inflammation, elevated total cell and eosinophil count, and altered cytokine levels in OVA-sensitized mice. These parameters improved upon inoculation with IDO-expressing imDCs. Co-culture with IDO-expressing imDCs also induced apoptosis, inhibited IL-4 and IL-5 expression, and upregulated IFN-gamma expression in CD4+ T cells. CONCLUSIONS: IDO-expressing imDCs induced T(H)2 cell apoptosis and reduced T(H)2 cell activation and allergic airway inflammation in OVA-sensitized mice. Thus, upregulation of IDO expression may provide a novel immunointervention strategy for asthma treatment.

Screening of mimetic peptides for CD14 binding site with LBP and antiendotoxin activity of mimetic peptide in vivo and in vitro.

OBJECTIVE AND DESIGN: The study was aimed at screening out the mimetic peptides from the binding site of lipopolysaccharide binding protein and CD 14, and then observing if the mimetic peptide will inhibit in vitro LPS-induced inflammatory reaction and function as an anti-endotoxin in the model of LPS-induced acute lung injury. MATERIAL AND METHODS: Human monocytic cell line (U937) was used in vitro. Thirty three-month-old SD rats were used. Phage display peptide library was adapted to screen mimetic peptide sequences. TREATMENT: U937 cells were exposed to treatment with LPS and rhLBP and then were incubated with MP12 at three different concentrations after they were induced and differentiated by PMA. LPS intravenous injection was used to establish a model of rat acute lung injury which was later treated with intravenous injection of MP12. RESULTS: We successfully obtained the mimetic peptide of lipopolysaccharide-binding protein and CD 14 binding site, the gene sequence of which is FHRWPTWPLPSP (MP12). MP12 can markedly inhibit LPS induced TNF-alpha expression. MP12 can evidently increase PaO(2) of rats with acute lung injury and also increase the survival rate of these rats. CONCLUSIONS: MP12 (FHRWPTWPLPSP) has the same function as mimetic of lipopolysaccharide-binding protein and CD 14 binding site. The application of MP12, both in vitro and in vivo, confers the biological activity required to antagonise LBP/CD14 and block LPS inflammatory signals, and it can markedly enhance PaO(2) of rats suffering from acute lung injury and also enhance their survival rate.

Killing effects of ganciclovir on human pulmonary adenocarcinoma cell A549 transduced with HSV1-TK gene in vitro and in vivo.

AIM: To observe the killing effects of ganciclovir (GCV) on the human pulmonary adenocarcinoma cell A549 transduced with Herpes simplex virus I type thymine kinase (HSV1-TK) gene in vitro and in vivo. METHODS: A retroviral vector containing the TK gene was constructed and transduced into a pulmonary carcinoma cell A549 by electroporation, to observe the sensitivity of the transfected cell to GCV in vitro and the bystander effect (MTT assay). Tumor cell apoptosis caused by the TK/GCV system was observed with a flow cell meter (FCM) and a scan electronic microscope (SEM). Recombination and expression of the TK gene were examined with DNA PCR and in situ hybridization, respectively. The therapeutic effect of GCV on subcutaneous tumor growth between transfected and parental cells was also compared. RESULTS: The sensitivity of the transfected cell to GCV was 46 times higher than that of the parental cell, and the bystander effect was stronger in high cell density than in low cell density. The subG0G1 peak was shown on the DNA histogram after A549-Tk cell was treated with 50 micromol/L GCV for 3 days by FCM, but not in the A549 cell. A cell cycle analysis showed that the apoptotic cell in the A549-TK and A549 cells were (12.2+/-1.7) % and (1.3 +/- 0.3) %, respectively (P < 0.01). The cell apoptosis features of nuclear condensation, apoptotic vesicle, and nuclear showing semimoon feature were found in the A549-TK cell by SEM, but not in the A549 cell. Recombination and expression of the TK gene were positive in the transfected cell. In vivo, the growth of tumors formed by the transfected cell was apparently inhibited by GCV, but not in the control group. CONCLUSION: The transfected cell obtained sensitivity to GCV and the bystander effect was closely related to intercellular touch. The TK/GCV system killing tumor cell was related to cell apoptosis. GCV inhibited the growth of tumors which were inoculated by A549-TK cell in vivo.

Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer.

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: results of urine genotoxicity assays as related to smoking habits.

A Phase II chemoprevention trial was carried out in Qidong, Jiangsu Province, People's Republic of China. The recruited subjects, all of whom were positive for serum aflatoxin-albumin adducts, were divided into three treatment arms: placebo; oltipraz ([5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione]) given daily at 125 mg p.o.; and oltipraz given once per week at 500 mg p.o. Besides biomarkers related to aflatoxin B(1) exposure, the genotoxicity of blind-coded urine XAD-2 concentrates was evaluated in 201 subjects on the fifth and seventh week of intervention. Genotoxicity was assessed both in the Ames reversion test in strain YG1024 of Salmonella typhimurium, in the presence of an exogenous metabolic system (S9 mix), with or without beta-glucuronidase, and in a DNA repair test in Escherichia coli. Heating of concentrated urine samples or of cigarette smoke condensates was discovered to result in a significant enhancement of their mutagenicity. It was also found that the mutagenicity of condensates from the most extensively used brands of cigarettes in Qidong was much lower than that of Western cigarette brands. Urine mutagenicity was unrelated to treatment with oltipraz, intervention time, gender, and supplement of S9 mix with beta-glucuronidase. Mutagenicity was significantly but variably higher in cigarette smokers than in nonsmokers, which suggests that the urinary excretion of mutagens in the examined population was not exclusively attributable to smoking. Nevertheless, within smokers (28% of the recruited subjects; 67% of all males), the mutagenic potency was significantly correlated with the self-reported number of cigarettes smoked per day and, even more sharply, with the cotinine concentrations in urines. In conclusion, this study demonstrated the validity of urine mutagenicity assays as a biomarker of tobacco smoke exposure that can be investigated on a relatively large scale in chemoprevention trials and provided evidence that oltipraz treatment had no influence on this parameter in the examined population.

Full-length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma.

In hepatitis B virus (HBV)-endemic countries, the majority of hepatocellular carcinoma (HCC) arises in HBV carriers. High frequency of mutations at nucleotides 1762(A-->T) and 1764(G-->A) in the core promoter region have been described in HCC. Due to the differences in genetic backgrounds, environmental risk factors and random cellular insertion sites, it is difficult to analyze the possible roles of HBV variants detected in different HCC patients. In a follow-up cohort study, an HBsAg-positive asymptomatic carrier was diagnosed HCC within 4 years. Eleven full-length HBV isolates, three from the first serum sample obtained 4 years pre-HCC, and eight from serum sample, peri-tumor and tumor tissue post-HCC of this individual were sequenced and used to transfect HepG2 cells. When sequences were compared between pre- and post-HCC isolates, no single mutation common to all post-HCC isolates that differed from pre-HCC isolates was found. Among all 11 isolates, there were 20 predicted amino acid substitutions shared by two or more post-HCC isolates. These were located in the S(5), X(4), core(4), polymerase(4), pre-S1(2) and pre-S2(1) proteins. Possible roles of amino acid substitutions and enhanced replication efficiency in cells transfected by post-HCC isolates are discussed.

Full-length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma.

In hepatitis B virus (HBV)-endemic countries, the majority of hepatocellular carcinoma (HCC) arises in HBV carriers. High frequency of mutations at nucleotides 1762(A-->T) and 1764(G-->A) in the core promoter region have been described in HCC. Due to the differences in genetic backgrounds, environmental risk factors and random cellular insertion sites, it is difficult to analyze the possible roles of HBV variants detected in different HCC patients. In a follow-up cohort study, an HBsAg-positive asymptomatic carrier was diagnosed HCC within 4 years. Eleven full-length HBV isolates, three from the first serum sample obtained 4 years pre-HCC, and eight from serum sample, peri-tumor and tumor tissue post-HCC of this individual were sequenced and used to transfect HepG2 cells. When sequences were compared between pre- and post-HCC isolates, no single mutation common to all post-HCC isolates that differed from pre-HCC isolates was found. Among all 11 isolates, there were 20 predicted amino acid substitutions shared by two or more post-HCC isolates. These were located in the S(5), X(4), core(4), polymerase(4), pre-S1(2) and pre-S2(1) proteins. Possible roles of amino acid substitutions and enhanced replication efficiency in cells transfected by post-HCC isolates are discussed.

SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.

Hepatocellular carcinoma (HCC) is a major cause of cancer death, but the molecular mechanism for its development beyond its initiation has not been well characterized. Suppressor of cytokine signaling (SOCS-1; also known as JAB and SSI-1) switches cytokine signaling 'off' by means of its direct interaction with Janus kinase (JAK). We identified aberrant methylation in the CpG island of SOCS-1 that correlated with its transcription silencing in HCC cell lines. The incidence of aberrant methylation was 65% in the 26 human primary HCC tumor samples analyzed. Moreover, the restoration of SOCS-1 suppressed both growth rate and anchorage-independent growth of cells in which SOCS-1 was methylation-silenced and JAK2 was constitutively activated. This growth suppression was caused by apoptosis and was reproduced by AG490, a specific, chemical JAK2 inhibitor that reversed constitutive phosphorylation of STAT3 in SOCS-1 inactivated cells. The high prevalence of the aberrant SOCS-1 methylation and its growth suppression activity demonstrated the importance of the constitutive activation of the JAK/STAT pathway in the development of HCC. Our results also indicate therapeutic strategies for the treatment of HCC including use of SOCS-1 in gene therapy and inhibition of JAK2 by small molecules, such as AG490.

Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry.

Hepatocellular carcinoma (HCC), a common cause of cancer deaths worldwide, has several major etiological risk factors, including infection with the hepatitis viruses and exposure to aflatoxin B1. A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B1. Short oligonucleotide mass analysis was compared with DNA sequencing in 25 HCC samples for specific p53 mutations. Mutations were detected in 10 samples by short oligonucleotide mass analysis in agreement with DNA sequencing. Analysis of another 20 plasma and tumor pairs showed 11 tumors containing the specific mutation, and this change was detected in six of the paired plasma samples. Four of the plasma samples had detectable levels of the mutation; however, the tumors were negative, suggesting possible multiple independent HCCs. Ten plasma samples from healthy individuals were all negative. This molecular diagnostic technique has implications for prevention trials and for the early diagnosis of HCC.

Hepatocellular carcinoma and aflatoxin exposure in Zhuqing Village, Fusui County, People's Republic of China.

Hepatocellular carcinoma (HCC) is a common cause of cancer morbidity and mortality in Asia and Africa. Epidemiological studies have found that dietary exposure to aflatoxin B1 (AFB1) and chronic infection with hepatitis B virus are two major risk factors for HCC. We have collated the incidence and mortality data of malignant tumors from 1973 to 1999 in Zhuqing Village, Fusui County, an area with very high HCC rates, and found that this cancer accounted for 64% of the total cancer incidence. Dietary intake of AFB1 was monitored for 1 week in a study group consisting of 15 males and 14 females from different households in this village. Four of 29 participants (13.8%) and 3 of 15 (20%) male participants were hepatitis B virus surface antigen positive. AFB1 was detectable in 76.7% (23 of 30) of ground corn samples (range, 0.4-128.1 ppb), 66.7% (20 of 30) of cooking peanut oil samples (range, 0.1-52.5 ppb), and 23.3% (7 of 30) of rice samples (range, 0.3-2.0 ppb) collected from each household. Mean levels of serum AFB1-albumin adducts in this group were 1.24 +/- 0.31 pmol/mg of albumin at the beginning of the study and 1.21 +/- 0.19 pmol/mg of albumin at the end of the period. Urinary AFB1 metabolites were detectable in 88.9% (24 of 27) samples (range, 0.9-3569.7 ng/24-h urine). These data provide the exposure and disease risk information for establishing intervention studies to diminish the impact of aflatoxin exposure in this high-risk population.

[Effect of Tripterygium wilfordii on Th1, Th2 cytokines production in asthma patients].

OBJECTIVE: To observe the effect of Tripterygium wilfordii on Th1, Th2 cytokines in asthma patients for further study on the therapeutic mechanism. METHODS: Twelve patients of middle or severe asthma were treated by Tripterygium polyglucoside 40 mg or 60 mg daily for 4 weeks. Blood of patients was colleted before and after treatment for serum and peripherol blood mononuclear cells (PBMC) preparation. The prepared PBMCs were stimulted in vitro with Concanavalin A (ConA) for 6 hrs and followed by culturing with Triptolide for 24 hrs and then the supernatant was collected. The concentration of interleukin-2(IL-2), -4(IL-4), -5(IL-5) and interferon-gamma(IFN-gamma) in serum and in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum levels of IL-2, IL-4 and IL-5 of patients decreased significantly after treatment of Tripterygium polyglucoside (P < 0.01), but IFN-gamma level was under the detection sensitivity both before and after treatment. Triptolide could inhibit PBMC to secrete IL-2, IL-4 and IL-5 in vitro (P < 0.01), but IFN-gamma was also under the detection sensitivity. CONCLUSION: The marked inhibition of Th2 cytokine expression by Tripterygium was the important mechanism of it in treating asthma. But the fact that Tripterygium also showed inhibition on Th1 cytokine indicated that the inhibition of Tripterygium on Th2 and Th1 cytokines was non-specific.

[Development and clinical application of the full automatic animal rearing cabin of low oxygen and high carbon dioxide].

This paper introduces a kind of automatic animal rearing cabin of low oxygen and high carbon dioxide. It can mimic the environment of low oxygen and high carbon dioxide at atmospheric pressure and automatically measure and control the concentrations of oxygen and carbon dioxide as well as temperature and humidity in the cabin. The system may provide the equipment support for clinical COPD study. The clinical applications show that the cabin with accurate measurement and control is practical and reliable.

[Changes in SR Ca2+-ATPase activity, Ca2+ release and uptake kinetics of diaphragm muscle after different-frequency chronic electrical stimulation of diaphragmatic nerve].

To study adaptation of rabbit diaphragm muscle after different-frequency chronic electrical stimulation, Ca(2+)-ATPase activity and Ca(2+) release-uptake kinetics of sarcoplasmic reticulum (SR) were respectively measured by detecting inorganic phosphorus ion and Furo-2 fluorescence. SR Ca(2+)-ATPase activity of the low-frequency stimulation group was significantly lower than that of the control group (P<0.0l), but it was significantly higher in the high-frequency stimulation group against control (P<0.0l). The kinetics of Ca(2+) release and Ca(2+) uptake was significantly lower in low-frequency group than that of the control (P<0.0l), but the kinetics of Ca(2+) release and Ca(2+) uptake was significantly higher than that of the control (P<0.01). It is thought that different-frequency electrical stimulation induced different adaptative changes in SR Ca(2+)-ATPase activity, and Ca(2+) release and uptake kinetics of rabbit diaphragm muscle.

[Experimental research on permeability injury of pulmonary microvascular endothelial cells monolayer induced by tumor necrosis factor].

AIM AND METHODS: To investigate the mechanism of tumor necrosis factor (TNF) induced permeability injury of rat pulmonary microvascular endothelial cells (RPMVEC) monolayer, the effect of TNF on permeability of RPMVEC monolayer was examined with microfilter and the effect of TNF on RPMVEC F-actin was observed with immunocytochemistry. The interfering action of formoterol, anisodamine and cholera toxin on permeability and F-actin changes induced by TNF was also observed. RESULTS: (1) TNF induced significant increase in permeability of RPMVEC monolayer 30, 60 and 90 minutes after treatment with TNF. (2) F-actin in RPMVEC depolymerized 90 minutes after treatment with TNF. Permeability and F-actin did not change significantly when formoterol, anisodamine or cholera toxin was added separately. CONCLUSION: TNF can induce permeability injury of RPMVEC monolayer, which is correlated with depolymerization of F-actin. Formoterol, anisodamine and cholera toxin can inhibit the permeability change induced by TNF which may due to their inhibition to the distribution change of F-actin.

[Adaptation of diaphragm muscle strip mechanics to chronic electrical stimulation and the effect of change in extracellular Ca2+ in rabbit].

AIM: The mechanical character of diaphragm muscle after chronic electrical stimulation(CES) and effect of the extracellular Ca2+ change have not yet been explored. We wondered whether there might be great different change and effect on muscle mechanics after CES and the extracellular Ca2+ change. METHODS: The twitch tension(Pt), time to peak tension(TPT), half-relaxation time(1/2 RT), tetanic tension(Po), fatigue index (FI) and fatigue recovery index(FRI) were respectively measured in normal group and CES groups; the switch tensions of diaphragm muscle strips were observed in the standard Hank's solution and the Hank's with free Ca2+. RESULTS: There were more significant decrease in Pt, Po, FI and FRI, more significant lengthening in TPT and 1/2 RT in 10 Hz and 20 Hz groups(P < 0.01). However, there was completely opposite effect in 50 Hz and 100 Hz groups. There were more significant effect on muscle mechanics of contraction and relaxation in 10 Hz and 20 Hz groups than that in 50 Hz and 100 Hz groups when the extracellular Ca2+ changed. CONCLUSION: After CES the significantly frequency dependent were presented on mechanical character of diaphragm muscle strips, and there were more effect on diaphragm muscle mechanics in 10 Hz and 20 Hz groups when the extracellular Ca2+ was changed.

[mRNA and protein expression of skeletal DHPR(alpha1) and RyRs in diaphragm muscle of rabbits].

To detect mRNA and protein expression of skeletal dihydropridine receptor isoform alpha1 subunit and ryanodine receptor 1 and 3 in diaphragm muscle of rabbits, the coupling mode and characteristics of Ca(2+) release were explored. Reverse transcription PCR, in situ hybridization and immunohistochemical methods were employed. A higher level of mRNA and protein expression of DHPR(alpha1) and RyR(1), and a lower level of mRNA expression of RyR(3) were found. It is suggested that the calcium release unit may consist of skeletal DHPR isoform, RyR(1) and RyR(3), and there may be two kinds of Ca(2+) release mode via conformational changes in linked proteins and Ca(2+)-induced Ca(2+) release (CICR) in diaphragm muscle of rabbits.

Elevated HPRT mutation frequencies in aflatoxin-exposed residents of daxin, Qidong county, People's Republic of China.

Molecular biomarkers are becoming increasingly important tools to identify people who are at highest risk of developing cancer. For many years we have been studying residents of Qidong County, People's Republic of China, to examine the combined impact of aflatoxin exposure with other risk factors as contributors to the high liver cancer incidence rates in this region. This study was conducted to determine the effects of aflatoxin exposure, as measured by serum aflatoxin-albumin adduct levels, on somatic mutation frequency in the human hypoxanthine guanine phosphoribosyl transferase gene (HPRT). Subjects were assigned as low or high according to a dichotomization around the population mean of aflatoxin-albumin adducts. HPRT mutant frequency was determined in individuals by a T cell clonal assay and the samples were categorized as low or high according to mean values. Separate analyses were also conducted for the small set of hepatitis B virus surface antigen (HBsAg)-positive and the larger set of HBsAg-negative individuals, known risk factors for liver cancer. An odds ratio of 19.3 (95% confidence interval 2.0, 183) was demonstrated for a high HPRT mutation frequency in individuals with high aflatoxin exposure compared with those with low aflatoxin exposure. This association indicates that aflatoxin-induced DNA damage in T lymphocytes, assessed using the validated surrogate albumin adduct markers, leads to increased mutations reflected as elevated HPRT gene mutations. This cross-sectional study suggests the potential use of mutation frequency of the HPRT gene as a long-term biomarker of aflatoxin exposure in high risk populations.

Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and p53 gene mutations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of HCC.

Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China.

BACKGROUND: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models. METHODS: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided. RESULTS: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682). CONCLUSIONS: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.