Mechanistic study of BNP7787-mediated cisplatin nephroprotection: modulation of human aminopeptidase N

Purpose

Previous studies from our laboratory have identified a role for gamma-glutamyl transpeptidase (GGT) in BNP7787 (disodium 2,2??-dithio-bis ethane sulfonate, dimesna, Tavocept?)-mediated cisplatin nephroprotection. Dekant has proposed that gamma-glutamyl transpeptidase (GGT), aminopeptidase N (APN) and cysteine-conjugate-??-lyase (CCBL) comprise a multi-enzyme pathway that acts on xenobiotic-glutathione conjugates converting them to nephrotoxic metabolites. We report modulation of APN activity within this pathway by BNP7787-derived mesna-disulfide heteroconjugates.

Methods

A fluorimetric assay was used to determine the effect of BNP7787, BNP7787-derived mesna-disulfide heteroconjugates, and the BNP7787 metabolite, mesna (sodium 2-mercaptoethane sulfonate), on the initial velocity and overall progress curve of the human APN reaction in vitro.

Results

Neither BNP7787 nor mesna-cysteinyl-glutamate inhibited human APN. Select BNP7787-derived mesna-disulfide heteroconjugates (mesna-cysteine, mesna-glutathione, mesna-cysteinyl-glycine) and high concentrations of mesna inhibited APN activity. Allosteric effects on the enzyme progress curve outside of the linear initial velocity region were observed for mesna-cysteinyl-glycine, mesna-glutathione and mesna-cysteinyl-glutamate and appeared to be a function of having both mesna and di- or tri-peptide functionalities in one molecule. In?situ-generated mesna-cisplatin conjugates were not a substrate for human APN.

Conclusions

BNP7787-mediated prevention or mitigation of cisplatin-induced nephrotoxicity may involve APN inhibition by certain BNP7787-derived mesna-disulfide heteroconjugates and appears correlated to the presence of a glycinate moiety and/or an anionic group. Two general mechanisms for BNP7787-mediated nephroprotection of cisplatin-induced nephrotoxicity involving the GGT, APN and CCBL nephrotoxigenic pathway are proposed which acting in a concerted and/or synergistic manner, and thereby prevent or mitigate cisplatin-induced renal toxicity.     

Human germ cell tumours: expression of gamma-glutamyl transpeptidase and sensitivity to cisplatin.

Previous studies have shown that the enzyme-glutamyl transpeptidase (GGT) is essential for the nephrotoxicity of cisplatin. This study was designed to determine whether GGT activity is necessary for the therapeutic effect of the drug. The relationship between GGT expression and clinical response to platinum-based chemotherapy was examined in 41 human germ cell tumours. Sections of formalin-fixed, paraffin-embedded tumours were immunohistochemically stained with an antibody directed against human GGT. There was no expression of GGT in any of the 17 seminomas or four dysgerminomas; whereas, 12/12 ovarian yolk sac tumours and 4/4 embryonal carcinomas of the testis were GGT-positive. In stage I tumours fewer tumour cells expressed GGT than in later stage tumours. In four germ cell tumours of mixed histology, the seminomatous and dysgerminoma areas were GGT-negative while the areas of the tumour with yolk sac or embryonal histology contained GGT-positive tumour cells. The patients with seminomas or dysgerminomas who were treated with cisplatin-based chemotherapy, all had a complete response despite the absence of GGT expression in these tumours. Fifteen of the 16 patients with yolk sac or embryonal carcinomas received cisplatin-based chemotherapy following surgery. Twelve had a complete response, while three failed to respond to platinum-based therapy. There was no correlation between the level of GGT-expression and response to therapy in this group. Three of the four patients with tumours of mixed histology were treated with cisplatin-based therapy, and had a complete response. Therefore, expression of GGT is not necessary for the therapeutic effect of cisplatin in germ cell tumours. The results from this study suggest that systemic inhibition of GGT would inhibit the nephrotoxic side-effect of cisplatin without interfering with its activity towards germ cell tumours.     

Modulation of plasma thiols and mixed disulfides by BNP7787 in patients receiving paclitaxel/cisplatin therapy

BACKGROUND: BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) was evaluated in a phase I clinical trial with paclitaxel and cisplatin to assess the safety and potential efficacy for preventing or reducing cisplatin- and paclitaxel-induced toxicities. During this trial the effects of BNP7787 administration on the total concentrations (oxidized plus free) of cysteine, homocysteine and GSH in plasma, free and total GSH in WBC and rate of urinary excretion of cysteine were studied. The pharmacokinetics of ultrafilterable (free, non-protein bound) platinum were also determined after cisplatin (75 mg/m(2)) treatment which followed paclitaxel (175 mg/m(2)) and BNP7787 (8.2 to 27.6 g/m(2)). METHODS: Plasma thiols were measured by HPLC with fluorescence detection and platinum was measured by atomic absorption spectrophotometry. RESULTS: BNP7787 administration produced a significant depletion of all plasma thiols in all the patients studied. Differences were noted in the kinetics of BNP7787-induced depletion of cysteine and other thiols. A significant depletion of cysteine occurred with a time lag of about 2 h after the end of BNP7787 infusion, while a reversible depletion of GSH and homocysteine occurred immediately following the start of BNP7787 infusion, with the plasma thiol/disulfide nadir corresponding to the end of infusion. The mean half-life of cysteine depletion following BNP7787 administration was 2.2 h, significantly longer than for homocysteine (0.23 h), or GSH (0.18 h; P<0.05 for both). A several-fold increase in the urinary excretion of cysteine occurred following BNP7787 administration in all patients. The BNP7787-induced thiol/disulfide depletion in plasma was not affected by cisplatin administration ( P>0.05). BNP7787 administration had no effect on the ultrafilterable platinum pharmacokinetics. The 2-h lag in the depletion of cysteine, the most abundant thiol in plasma, suggests that the process may be related to the formation of free mesna from BNP7787 and that increased levels of mesna are not in circulation until after 2 h after BNP7787 administration. No effect of BNP7787 was seen on the GSH concentration in WBC, possibly reflecting the inability of these cells to take up BNP7787. CONCLUSION: The results suggest that BNP7787 has the potential to enhance cisplatin antitumor activity by depleting the reactive thiols in plasma.     

Phase I and pharmacokinetic study of the novel chemoprotector BNP7787 in combination with cisplatin and attempt to eliminate the hydration schedule

BNP7787 (disodium 2,2'-dithio-bis-ethane sulphonate; Tavocept) is a novel agent developed to protect against cisplatin (cis-diammine-dichloroplatinum(II))-associated chronic toxicities. In this study, we determined the recommended dose of BNP7787 when preceding a fixed dose of cisplatin, the pharmacokinetics (PKs) and the possible reduction of saline hydration. Patients with advanced solid tumours received BNP7787 in escalating doses of 4.1-41 g m(-2) as a 15-min intravenous (i.v.) infusion followed by cisplatin 75 mg m(-2) as a 60-min i.v. infusion together with pre- and postcisplatin saline hydration in a volume of 2200 ml; cycles were repeated every 3 weeks. PK was carried out using BNP7787, cisplatin and the combination. Twenty-five patients were enrolled in stage I of the study to determine the recommended dose of BNP7787. No dose-limiting toxicity was reached. The highest dose level of 41 g m(-2) resulted in a low incidence of grade 2 toxicities, being nausea and vomiting, dry mouth or bad taste and i.v. injection site discomfort. Doses of BNP7787 > or = 18.4 g m(-2) did not show a drug interaction between BNP7787 and cisplatin. In stage II of the study, patients received a fixed dose of BNP7787 of 18.4 g m(-2) preceding cisplatin and were entered in prespecified reduced saline hydration steps. A total of 21 patients in cohorts of six to nine patients received reduced saline hydration of 1600 ml (step A), 1000 ml (step B) and 500 ml (step C). In step C, two out of six evaluable patients experienced grade 1 nephrotoxicity. Cisplatin acute toxicities in all 46 patients were as expected. Only five patients complained of paresthesias grade 1 and six developed slight audiometric changes. Partial tumour response was observed in four patients and stable disease in 15 patients. In conclusion, BNP7787 was tolerated well up to doses of 41 g m(-2). The recommended dose of 18.4 g m(-2) enabled safe reduction of the saline hydration schedule for cisplatin to 1000 ml. Further studies will assess whether BNP7787 offers protection against platinum-related late side effects.     

BNP7787, a novel protector against platinum-related toxicities,does not affect the efficacy of cisplatin or carboplatin in human tumour xenografts

BNP7787 (2',2'-dithio-bis-ethane sulphonate sodium), a water-soluble disulphide, is chemically and mechanistically different from other sulphur-containing chemoprotective agents. Presently, BNP7787 is under investigation for its protective properties with regard to the side-effects of platinum compounds. In this study, we evaluated BNP7787, mesna and amifostine for their effects on the antitumour activity of platinum compounds. Continuous exposure to BNP7787 did not affect the antiproliferative effects of cisplatin or carboplatin, but the efficacy of both compounds was reduced in the presence of mesna in vitro in two human ovarian cancer cell lines. BNP7787 or amifostine combined with cisplatin or carboplatin given in standard schedules for the treatment of nude mice bearing well-established OVCAR-3 xenografts did not interfere with platinum-induced inhibition of tumour growth. Of interest, BNP7787 or amifostine co-administered with carboplatin was significantly more effective than carboplatin alone (P<0.01). In the presence of amifostine, doses of cisplatin and carboplatin could be safely increased by factors of 1.6 and 1.5, respectively. Unlike in a previous study of BNP7787 in tumour-bearing rats, BNP7787 did not protect against additional weight loss following treatment with higher doses of cisplatin in OVCAR-3-bearing mice. Pharmacokinetics of (mixed) disulphides including BNP7787 and extractable mesna in deproteinised plasma revealed a rapid disappearance of BNP7787 and an AUC(5-60) value of mesna 9-fold lower than that calculated after an equivalent dose of mesna by weight. We can conclude that BNP7787 does not interfere with the antitumour activity of platinum compounds in vitro and in vivo. Clinical trials are underway to evaluate the protection of normal tissues by BNP7787 when combined with cisplatin.     

Pharmacokinetics of BNP7787 and its metabolite mesna in plasma and ascites: a case report

PURPOSE: BNP7787 (2',2'-dithio-bis-ethane sulfonate sodium) is a novel protector against cisplatin-induced toxicities. The pharmacokinetics of BNP7787 and its metabolite mesna were investigated in plasma and ascites of a cancer patient. We also evaluated potential pharmacokinetic interactions between BNP7787 and cisplatin. METHODS: BNP7787 and mesna were measured as mesna in deproteinized plasma and ascites using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. RESULTS: After the i.v. administration of 41 g/m(2) BNP7787, BNP7787 and mesna had a half-life of 1.5 and 3.4 h, respectively. The AUC( infinity ) of mesna was approximately 8% of the AUC( infinity ) of BNP7787. Coadministration of cisplatin did not appear to influence the plasma concentration-time curves of BNP7787 and mesna. In ascites, approximately 0.02% of the BNP7787 dose was present as mesna, whereas approximately 4% of the dose was present as BNP7787 at the time of the maximum concentration. CONCLUSIONS: It can be concluded that the presence of ascites did not have a major impact on the pharmacokinetics of BNP7787 and coadministration of cisplatin did not influence the pharmacokinetics of BNP7787 and mesna.     

Expression of gamma-glutamyl transpeptidase during pouch carcinogenesis is not oncofetal

gamma-glutamyl transpeptidase (GGT) is not histochemically detectable in normal adult hamster pouch epithelium but markedly increased during pouch carcinogenesis. The possibility that the increase in GGT activity during pouch carcinogenesis reflects dedifferentiation of cells remains to be answered. The distribution of GGT in fetal and neonatal hamster tissues is unknown. The objective of this study was to determine GGT activity histochemically during hamster pouch development and during development of hamster oral and peri-oral structures, and several extra-oral tissues and organs. High GGT activity was seen mainly in epithelial cells exhibiting marked secretory or absorptive functions. Occasionally, GGT activity was also seen in mesenchymal cells. No GGT activity was noted at any stage of pouch development. The results suggest that the expression of GGT during pouch carcinogenesis represents an acquired phenotype instead of re-expression of a phenotype that is present during normal embryonic development. The results also support the hypothesis that GGT plays an important role in transportation.     

Phase I and pharmacologic study of BNP7787, a novel chemoprotector in patients with advanced non-small cell lung cancer

Purpose

We conducted a phase I trial of BNP7787 (disodium 2,2??-dithio-bis-ethane sulfonate, Tavocept?), a novel chemoprotective and antitumor enhancing agent administered in combination with paclitaxel and cisplatin. The primary aim was to determine a safe and potentially efficacious BNP7787 dose for preventing and mitigating paclitaxel- and cisplatin-induced toxicities and to evaluate for preliminary evidence of efficacy of treatment.

Patients and methods

Twenty-two patients with stage IIIB/IV non-small cell lung cancer (NSCLC) received BNP7787 alone 1 week before co-administration of BNP7787 with paclitaxel followed by cisplatin. Twenty-one patients were treated with BNP7787 in escalating doses of 4.1?C41.0?g/m² concurrently with paclitaxel 175?mg/m² and cisplatin 75?mg/m² every 3?weeks.

Results

The appropriate dose was determined to be 18.4?g/m² of BNP7787 although no dose-limiting toxicity was observed up to 41.0?g/m². Mild intravenous site discomfort, thirst, and nausea were the most common toxicities. One patient developed grade 2 skin rash, which was severe enough to preclude further study treatment. The AUC_0-inf of the metabolite mesna was approximately 6.3% of the AUC_0-inf of BNP7787. Co-administration of paclitaxel and cisplatin did not appear to influence the pharmacokinetics of BNP7787 and mesna. The overall response rate was encouraging; 43% including 11 patients with prior chemotherapy.

Conclusions

The recommended dose for phase II/III studies is 18.4?mg/m² of BNP7787 in combination with paclitaxel and cisplatin. Further studies are warranted to assess whether BNP7787 prevents and mitigates common and serious paclitaxel- and cisplatin-related side effects and enhances the efficacy of paclitaxel and cisplatin in advanced NSCLC patients.     

The chemical reactivity of BNP7787 and its metabolite mesna with the cytostatic agent cisplatin: comparison with the nucleophiles thiosulfate,DDTC, glutathione and its disulfide GSSG

PURPOSE: BNP7787 is a new chemoprotective agent presently under clinical investigation to protect against cisplatin-induced toxicities, especially nephrotoxicity and neurotoxicity. In the kidneys BNP7787 is postulated to undergo selective conversion into mesna, which can locally detoxify cisplatin. The reactivity of cisplatin with this new chemoprotective agent and with its metabolite mesna was investigated at clinically observed plasma concentrations and compared with the nucleophiles thiosulfate (TS) and DDTC, and with the endogenous compounds glutathione (GSH) and oxidized glutathione (GSSG). METHODS: Reaction kinetics experiments were performed at 37 degrees C and pH 7.4 in the presence of a high chloride concentration (0.15 M). The degradation of cisplatin was measured over time using HPLC with off-line flameless atomic absorption spectrophotometry. RESULTS: The degradation half-lives of cisplatin (13.5 microM) with 17.2 m M BNP7787, 340 microM mesna and 17.2 m M mesna were 124 min, about 790 min and 73 min, respectively. Cisplatin reacted at least 9.5 times more slowly with 17.2 mM BNP7787 and 5.5 times more slowly with 17.2 mM mesna than with 17.2 mM of the modulating agents DDTC or TS (i.e. half-lives 11 and 13 min, respectively). The half-lives of cisplatin with 17.2 m M GSH and GSSG (i.e. 122 and 115 min, respectively) were comparable with the half-life obtained with BNP7787. The thiol mesna was shown to be a stronger nucleophile than its corresponding disulfide BNP7787. CONCLUSIONS: The much slower relative reactivity of BNP7787, the short residence of BNP7787 (approximately 2 h) and the much lower concentration of mesna in the circulation following BNP7787 administration precludes chemical inactivation of cisplatin in the circulation, and thus the antitumor activity of cisplatin is maintained.     

Effect of hexachlorobenzene on male and female rat hepatic gamma-glutamyl transpeptidase levels

The effect of 200 ppm hexachlorobenzene (HCB) in the diet on hepatic gamma glutamyl transpeptidase (GGT) levels was examined in F344 male and female rats. During early stages of feeding (4-29 weeks) some females had elevated GGT levels spread throughout the periportal regions, but all those examined at 29 weeks had a few GGT positive foci. The livers of males were not affected at this time. However, after 90 weeks of feeding, males also had elevated periportal GGT activity and a number of GGT positive foci. All females had lost the periportal activity but had developed GGT positive hepatocellular tumours. It therefore appeared that elevated nonfocal GGT levels are a poor indicator for HCB toxicity, but GGT positive foci may be a more reliable indicator of carcinogenic changes.     

Peroxisome proliferator-induced hepatocarcinogenesis: histochemical analysis of ciprofibrate-induced preneoplastic and neoplastic lesions for gamma-glutamyl transpeptidase activity

Previous studies revealed that putative preneoplastic and neoplastic lesions induced in the liver by Wy-14,643, a peroxisome proliferator, were gamma-glutamyl transpeptidase (GGT) negative. For ascertainment as to whether phenotypes of foci and carcinomas induced by all peroxisome proliferators are similarly GGT negative, altered areas (AAs), neoplastic nodules (NNs), and hepatocellular carcinomas (HCCs) induced in the livers of male F344 rats by chronic dietary administration of ciprofibrate (0.025% wt/wt in chow; CAS: 52214-84-3) were analyzed histochemically for GGT activity. Eighty-nine percent of AAs, 91% of NNs, and 91% of HCCs were GGT negative. The GGT-negative property of these various hepatic preneoplastic and neoplastic lesions persisted at 8 weeks after the withdrawal of ciprofibrate treatment. The results of this study indicate that the absence of GGT activity is a common feature in hepatic lesions induced by structurally unrelated peroxisome proliferators and is not related to the drug toxicity. The proposal was made that peroxisome proliferators do not derepress the activity of the GGT gene during hepatocarcinogenesis in the rat.     

The clonal nature of carcinogen-induced altered foci of gamma-glutamyl transpeptidase expression in rat liver

The clonality of tumors has been convincingly established. Because it is generally accepted that tumor formation involves a number of steps, it is important to determine which if any of the precursors of tumors are clonal. A series of chimeric rats produced between congenic strains by morulae aggregation were used to establish the cellular composition of foci of gamma-glutamyl transpeptidase (gamma-GTP; E.C. 2.3.2.2) expression in liver following initiation with N-nitrosodiethylamine and promotion with phenobarbital. The chimeras were produced between congenic rat strains (PVG and PVG-RT1a) genetically distinguished by alleles of the major histocompatibility complex (MHC). Monoclonal antibodies directed to distinctive class I MHC alloantigens were used to detect patterns of mosaicism in the animals. The parental genotypes present in most visceral tissues could be easily distinguished by our method. Analysis of 499 enzyme-altered foci revealed that 474 were comprised solely of either PVG-RT1a or PVG cells. Some apparent mixture of cells from the two lineages was observed in 25 lesions, most of which were very small. The observed pattern of distortion of normal patch distribution clearly indicated the expanding and clonal nature of these lesions.     

Immunofluorescent analysis of gamma-glutamyl transpeptidase and glutathione-S-transferase-P during the initial phase of experimental hepatocarcinogenesis

We used double-label immunofluorescence to evaluate the expression of glutathione-S-transferase-P (GST-P) and gamma-glutamyl transferase (gamma GT) in individual liver cells in rats placed on a modified Solt-Farber hepatocarcinogenesis protocol. Four days after treatment with diethylnitrosamine (DEN), single GST-P+ cells were found in the centrilobular and midzonal regions. Most of these were gamma GT-, but the percentage of gamma GT+ cells increased with DEN dose to a high of 32%. At later times (7-8 days) doublets and small clusters of GST-P+ cells predominated; all cells in each pair or cluster displayed the same phenotype (GST-P+ or GST-P+/gamma GT+). Following administration of acetylaminofluorene and partial hepatectomy, lesions were larger and a greater percentage were GST-P+/gamma GT+. However, within these lesions individual cells displayed different phenotypes. These results indicate that the expression of GST-P and gamma GT is discordant in individual cells and small clusters soon after treatment, and suggest that different molecular events may be involved in their expression.     

检测γ-谷氨酰转移酶mRNA亚型在肝癌早期诊断中的临床价值

背景及目的：与正常肝脏相比,肝癌细胞表达的γ－谷氨酰转移酶（GGT）有一独特的碳水化合物结构,这种GGT可作为肝癌诊断的重要指标。但是,在癌变过程中GGT基因的改变机制尚不清楚,本研究探讨GGTmRNA亚型的转化与原发性肝癌（HCC）发生的关系,寻找肝癌早期诊断的新方法。方法：以RT－PCR方法检测正常对照组,非癌肝病组,肝癌组及肝转移癌肝组织及外周血的三种GTTmRNA亚型（F,H,P亚型）。结果：正常肝组织,非癌肝组织及肝转移癌癌周组织主要的GGTmRNA类型为F亚型,肝癌组织,癌旁组织,及远离组织GGTmRNA-H亚型的阳性率显著高于正常肝脏及非癌肝病肝组织（P＜0．05）,肝癌组织GGTmRNA-F亚型阳性率明显低于正常对照及非癌肝病组（P＜0．05）。在26例HCC眙有12例外周血中检出GGTmRNA-H亚型,AFP阴性的10例HCC中有5例检出GGTmRNA－H亚型。结论：GGTmRNA亚型转化与肝癌发生有密切关系,分析GGT基因可望成为监测肝细胞癌变的灵敏方法。     

Expression of gamma-glutamyl transpeptidase in adult rat liver cells after transformation with SV40 virus

By the use of histochemical techniques we have shown that the transformation of rat hepatocytes with simian virus 40 resulted in a large majority of cells producing gamma-glutamyl transpeptidase (gamma GT). Using a temperature-sensitive mutant of this virus we have shown that the percentage of cells exhibiting this enzyme in a temperature-sensitive SV40 mutant is much higher in cells grown at the permissive temperature of 33 degrees C than at the nonpermissive temperature of 40.5 degrees C. We thus conclude that, the correlation between gamma GT expression and chemical or spontaneous transformation of hepatocytes, previously described by other authors, can now be extended to viral transformation and that the enzyme activity is dependent on the expression of a transformed cell phenotype.     

Pharmacokinetic behaviour of the chemoprotectants BNP7787 and mesna after an i.v. bolus injection in rats

In preclinical studies, BNP7787 (disodium 2,2'-dithio-bis-ethane sulphonate), the disulphide form of mesna, has demonstrated selective protection against cisplatin-induced nephrotoxicity due to conversion into mesna inactivating toxic platinum species. Mesna (sodium 2-mercapto ethane sulphonate), however, can affect the antitumour activity of cisplatin, while BNP7787 does not interfere with the antitumour activity. To understand the difference in interference with cisplatin-induced antitumour activity between BNP7787 and mesna as well to characterise the selective nephroprotection by BNP7787, the pharmacokinetics of BNP7787 and mesna, each given i.v. 1000 mg x kg(-1), were determined in plasma, kidney, liver, red blood cells (RBC), skeletal muscle and tumour of Fischer rats bearing subcutaneously implanted WARD colon tumours. The following results were obtained: (1). high concentrations of BNP7787 and mesna were observed in the plasma and kidney after administration of BNP7787 or mesna, except for mesna in plasma after BNP7787 administration; (2). in all other sampled compartments, the AUC values of both compounds were at least 5.5-fold lower than the corresponding values in kidney; (3). the AUC of mesna in plasma after mesna administration was comparable to the AUC of mesna in kidney after a dose of BNP7787 that can completely prevent cisplatin-induced nephrotoxicity in rats; (4). the AUC of mesna in plasma was five-fold higher relative to the AUC of mesna following BNP7787 administration (P<0.01). In conclusion, the five-fold higher AUC of mesna in plasma after mesna administration and the fact that mesna is more reactive with (hydrated) cisplatin than its disulphide form BNP7787 represent a plausible explanation as to why mesna administration can reduce the antitumour activity of cisplatin. After BNP7787 administration, the distribution of BNP7787 and mesna was restricted to the kidney, which confirmed the selective protection of the kidney by BNP7787.     

谷氨酰转肽酶与血小板比值在评估肝细胞癌肝纤维化分期中的价值

目的 探讨谷氨酰转肽酶与血小板比值（gamma-glutamy transpeptidase to platelet ratio,GPR）在评估肝细胞癌（hepatocellular carcinoma,HCC）患者肝纤维化分期中的价值。方法 收集2015年8月至2016年8月在广西医科大学附属肿瘤医院肝胆外科行肝切除术的HCC患者的临床资料,计算GPR、天门冬氨酸氨基转移酶与血小板比值指数（amino- transferase to platelet ratio index,APRI）、FIB4指数（index based on the 4 factors,FIB4）,用ROC曲线下面积比较GPR、APRI、FIB4诊断肝纤维化不同分期（S0期、S1期、S2期、S3期、S4期）的效能。结果 GPR、APRI、FIB4、谷丙转氨酶（alanine transaminase,ALT）与肝纤维化分期呈正相关（P＜0.05）,而血小板计数（blood platelet count,PLT）与肝纤维化分期呈负相关（r=-0.360,P＜0.001）;肝纤维化不同分期（S0期、S1期、S2期、S3期、S4期）中,GPR、APRI、FIB4、PLT值差异均有统计学意义（P＜0.05）;肝纤维化＞S0期,GPR曲线下面积分别与APRI、FIB4、PLT曲线下面积比较,差异均无统计学意义（P＞0.05）;肝纤维化＞S1期和＞S3期中,GPR曲线下面积分别相应与APRI、PLT曲线下面积比较,差异均无统计学意义（P＞0.05）,但高于FIB4曲线下面积,差异有统计学意义（P＜0.05）;肝纤维化＞S2期,GPR曲线下面积分别相应与APRI、PLT曲线下面积比较,差异均有统计学意义（P＜0.05）。结论 GPR诊断肝纤维化分期具有较高的准确性,可作为评估HCC患者术前肝纤维化分期的无创性指标。     

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.