Biomedical subjects

A M Rofe

Publications and source records attributed to A M Rofe.

At least 37 records · Page 2Linked to original sources

PubMed1998

Paracetamol hepatotoxicity in metallothionein-null mice.

The role of metallothionein (MT) in protecting the liver against paracetamol (PCT) toxicity was investigated in vivo and in vitro in mice lacking expression of MT-1 and MT-2 genes (MT -/-). In the fed, glycogen replete state, hepatotoxicity (PCT 300 mg/kg i.p.) at 6 h was significantly greater in MT -/- than MT +/+ mice. Plasma lactate dehydrogenase (LD) and alanine aminotransferase (ALT) were 5- and 13-fold greater respectively than in MT +/+ mice. Liver glycogen, glucose and zinc levels were significantly lower in MT -/- mice at this time. In contrast, hepatotoxicity (PCT 135 mg/kg i.p.) at 6 h was similar in both MT +/+ and MT -/- mice fasted 24 h, despite a doubling in liver MT in MT +/+ mice. No differences were found between MT -/- and MT +/+ mice in cytochrome P450 activity. Liver glutathione levels were the same in both groups of mice prior to fasting and were decreased to a similar extent (55-65%) following PCT treatment. Investigation of lower PCT doses (< or = 120 mg/kg) in fasted mice over 24 h demonstrated a greater susceptibility in female MT -/- mice with plasma LD, 2.4-fold and ALT, 7.5-fold greater than in MT +/+ mice at 120 mg/kg PCT. In male MT -/- mice, there was only a trend towards greater susceptibility at 110 mg/kg PCT compared to male MT +/+ mice, and at 120 mg/kg, both male genotypes were equally affected. Investigations with cultured hepatocytes supported the in vivo findings in that there was a trend towards greater toxicity (PCT at 1 and 5 mM for 24 h) in hepatocytes from fed MT -/- mice, with the difference diminished in association with greater hepatotoxicity in hepatocytes from fasted mice. Use of dexamethasone (Dex) to increase MT in the MT +/+ mouse hepatocytes protected from PCT toxicity. Zn alone was not protective. Zn plus Dex offered no protection despite higher MT levels. Generation of apo-MT with Dex may offer more protection than Zn-MT. In conclusion, MT -/- mice were more susceptible than MT +/+ mice to PCT toxicity in the fed state, but the increased susceptibility was much smaller, but still significant, when the effects of glycogen were minimised by fasting.

Acetaminophen↗

PubMed1998

Efficacy of cytotoxic agents for the prevention of laparoscopic port-site metastases.

BACKGROUND: Recent experimental studies support initial clinical impressions that laparoscopic surgery for malignant neoplasms may be associated with an increased incidence of metastases to port sites. This study investigated in an experimental model the influence of cytotoxic agents (administered intraperitoneally or intramuscularly) on the development of port-site metastases following laparoscopic surgery. METHODS: Seven days after the implantation of an adenocarcinoma in the left abdominal flank, 72 Dark Agouti rats underwent laparoscopy with carbon dioxide insufflation, instillation of an intraperitoneal agent, and intraperitoneal tumor laceration within the following study groups (12 rats in each group): (1) control (no intraperitoneal instillation); (2) intraperitoneal instillation of isotonic sodium chloride solution (0.9%); (3) intraperitoneal instillation of povodine-iodine (1:10 dilution of povidine-iodine and isotonic sodium chloride solution); (4) intraperitoneal instillation of methotrexate (0.125 mg of methotrexate in 3 mL of isotonic sodium chloride solution); and (5) intraperitoneal instillation of aqueous chlorhexidine acetate. Twelve additional rats underwent laparoscopic tumor laceration following intramuscular injection of 0.125 mg of methotrexate (no intraperitoneal agent). Rats were killed 7 days after the procedure, and the wounds were examined histologically by a blinded histopathologist for the presence of tumor metastases. RESULTS: No tumor was found in any port site following the intraperitoneal administration of povidine-iodine (P=.04). In contrast, port-site metastases developed in the control group (5 [41.7%] of 12), the isotonic sodium chloride solution group (4 [33.3%] of 12), the chlorhexdine group (4 [33.3%] of 12), the intraperitoneal methotrexate group (2 [16.7%] of 12), and the parenteral methotrexate group (5 [41.7%] of 12). CONCLUSIONS: The results of this study suggest that the development of metastases to port sites following laparoscopic surgery may be prevented by the intraperitoneal instillation of diluted povodine-iodine. Other agents failed to influence the incidence of port-site metastases. Further studies are needed to determine if these findings can be applied to humans.

Animals↗

PubMed1998

Regional distribution of metallothionein and zinc in the mouse gut: comparison with metallothionien-null mice.

Gut Zn homeostatic responses to low, replete, and excess dietary Zn (10, 150, and 400 mg Zn/kg, respectively) were compared in mice with (MT+/+) and without (MT-/-) metallothionein (MT) expression. MT concentrations decreased progressively from stomach (12.9 nmol Cd bound/g) to colon (4.6 nmol Cd bound/g). Small intestinal MT was increased in mice fed the 400-mg Zn/kg diet (+130%, duodenum; +56%, jejunum; +29%, terminal ileum), but not in the stomach, cecum and colon. Zn concentrations were much higher in the distal gut at increasing Zn intakes in MT+/+ mice but to a lesser extent in MT-/- mice. On the 10-mg Zn/kg diet, MT-/- mice had 45% more Zn in the jejunum/ileum than MT+/+ mice. In fasted (20 h) mice, Zn concentrations in all gut regions were similar to those of MT+/+ mice fed the 10-mg Zn/kg diet, irrespective of prior Zn intake or genotype. Liver MT quadrupled in mice fasted after the 10-mg Zn/kg diet but only doubled after the 400-mg Zn/kg diet, a trend also present in gut MT. Glucagon administration stimulated gut as well as liver MT, implicating it as a major component of the MT response to fasting. MT-/- mice had five times more variation than MT+/+ mice in plasma Zn over all dietary groups. Together, these findings demonstrate that without MT, there is little modification of regional gut Zn concentrations in response to extremes of dietary Zn and poorer regulation of Zn homeostasis.

Animals↗

PubMed1998

Tumor implantation following laparoscopy using different insufflation gases.

BACKGROUND: Laparoscopic manipulation of malignancies is associated with an increased incidence of metastasis to port sites in experimental models. This study investigated the effect of different insufflation gases on the implantation of a tumor cell suspension following laparoscopic surgery in an established small animal model. METHODS: Forty Dark Agouti rats underwent laparoscopy and the introduction into the peritoneal cavity of a tumor cell suspension. The insufflating gas used for each procedure was one of the following gases (10 rats in each group): carbon dioxide (CO2), nitrous oxide (N2O), helium, and air. The rats were killed 7 days after surgery, and the peritoneal cavity and port sites were examined for the presence of tumor. RESULTS: Although no significant differences were seen between air, CO2, and N2O insufflation groups, tumor involvement of peritoneal surfaces was less likely following helium insufflation. CONCLUSION: The results of this study suggest that tumor metastasis to port sites following laparoscopic surgery may be influenced by the choice of insufflation gas. In this study, helium was associated with reduced tumor growth.

Adenocarcinoma↗

PubMed1998

The effect of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) on urinary oxalate excretion: studies using a hyperoxaluric rat model.

PURPOSE: To determine the efficacy of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) in reducing urinary oxalate excretion under hyperoxaluric conditions and to determine whether by inclusion of glycolate in a standard diet, cysteine:glyoxylate adduct can be detected in hyperoxaluric rats given either compound. MATERIALS AND METHODS: Hyperoxaluria (200% above basal) was induced 2 days prior to commencement of the studies and maintained throughout. After a 3 days baseline, animals were randomly allocated to a control or treatment group. Standard diet containing either (L)-cysteine or OTZ was then fed to the treatment groups for 5 days while standard diet alone was fed to the control groups. Urinary oxalate excretion was subsequently monitored and average daily rates were then compared with basal values. Plasma and urine were analyzed for adduct. RESULTS: Both (L)-cysteine and OTZ significantly reduced urinary oxalate excretion relative to the basal hyperoxaluric level (28.6 +/- 1.5 micromol./day). While (L)-cysteine reduced oxalate excretion over the 5 day treatment period by only 7.82 +/- 1.39 micromol./day (27%), OTZ reduced it by 12.34 +/- 1.58 micromol./day (43%). Adduct could not be detected in plasma or urine in this study. CONCLUSIONS: This study confirms that both (L)-cysteine and OTZ are effective in reducing urinary oxalate excretion under hyperoxaluric conditions, with OTZ being more effective than (L)-cysteine. These compounds were shown to be 3- to 4-fold more effective in reducing urinary oxalate excretion under hyperoxaluric conditions when compared with the results from previous studies under normooxaluric conditions.

Animals↗

PubMed1998

Wound metastasis after laparoscopy with different insufflation gases.

BACKGROUND: There is growing evidence that laparoscopy for malignancy is associated with an increased incidence of metastasis to port sites. This study investigated the effect of different insufflation gases on port-site metastasis after laparoscopy in an established animal model. METHODS: Forty-eight Dark Agouti rats with an established adenocarcinoma in the left flank underwent laparoscopic intraperitoneal tumor laceration. The gas used for insufflation was one of the following (12 rats in each group): (1) CO2, (2) N2O, (3) helium, or (4) air. Rats were killed 7 days after the procedure, and the port sites were examined for the presence of tumor metastasis. RESULTS: Tumor involvement of port sites was significantly less likely after helium insufflation than in the other groups (p < 0.0001). There was no significant difference between the air, CO2, and N2O groups. CONCLUSIONS: This study suggests that the development of metastases in port sites after laparoscopy may be influenced in part by the choice of insufflation gas used to create the pneumoperitoneum. In particular, helium was associated with a reduced rate of metastases.

Adenocarcinoma↗

PubMed1997

Gasless laparoscopy may reduce the risk of port-site metastases following laparascopic tumor surgery.

OBJECTIVE: To compare the incidence of port-site metastases in an experimental tumor model following tumor manipulation during laparoscopy aided by conventional insufflation with laparoscopy using a gasless technique. SETTING: An experimental model applied in a research laboratory. PARTICIPANTS AND INTERVENTIONS: Malignant tumors were implanted in the abdominal wall of 24 rats. Twelve rats underwent tumor laceration at laparoscopy with carbon dioxide insufflation, and 12 rats underwent the same procedure during gasless laparoscopy achieved by abdominal wall suspension. Rats were killed 1 week later and were examined for evidence of tumor metastases. The surgical wounds were examined microscopically by a histopathologist who was unaware of the operative technique used and the site of origin of the specimens. MAIN OUTCOME MEASURE: Histologically confirmed tumor metastasis to laparoscopic port wounds. RESULTS: Growth of the primary tumor was equal in both groups. Wound metastases were less likely in the gasless laparoscopy group (3 of 12 vs 10 of 12; P = .01, Fisher exact test). CONCLUSION: The use of laparoscopy without gas insufflation may reduce the risk of wound metastasis following laparoscopic surgery for cancer.

Animals↗

PubMed1997

The effect of laparoscopy on the movement of tumor cells and metastasis to surgical wounds.

BACKGROUND: A variety of mechanisms have been proposed to explain tumor growth in port sites following laparoscopic cancer surgery. We devised two experimental models to determine whether carbon dioxide (CO2) insufflation during laparoscopic surgery influences the movement of tumor cells and leads to tumor implantation and growth in surgical wounds. METHODS: Model 1: Viable adenocarcinoma cells were introduced into the upper abdomen of six syngeneic immune-competent rats during laparoscopy with CO2 insufflation; the same procedure was followed for a further six rats during gasless laparoscopy. A length of plastic tubing introduced through the anterolateral aspect of the rats' left lower abdominal wall was used to vent the insufflation gas through the abdomen of a recipient rat for 30 min. After 21 days, the peritoneal cavity and surgical wounds of the recipient rat were examined for implanted tumor. Model 2: A suspension of radiolabeled adenocarcinoma cells was introduced into the upper abdomen of five rats during laparoscopy with CO2 insufflation and an additional five rats during gasless laparoscopy. A length of plastic tubing introduced through the anterolateral aspect of the left lower abdominal flank was used to vent the insufflation gas through phosphate-buffered saline solution. After 30 min, the solution was counted for radioactivity. RESULTS: Tumor growth occurred at the site of both the insufflation and venting ports in the second rat in five of the six rats from the group undergoing insufflation, but it was found in none of the gasless laparoscopy group (p = 0.015). In the second model, significant transfer of tumor cells to the vented gas occurred only in the rats undergoing laparoscopy with insufflation (median, 2.71% versus 0% of the introduced labeled cells; p = 0.008). CONCLUSIONS: Carbon dioxide insufflation results in tumor dissemination during laparoscopy, leading to port site metastasis. Gasless laparoscopy may prevent this problem.

Abdominal Muscles↗

PubMed1997

Adverse impact of pneumoperitoneum on intraperitoneal implantation and growth of tumour cell suspension in an experimental model.

BACKGROUND: An investigation of the effect of laparoscopy and CO2 pneumoperitoneum on the pattern of tumour implantation and growth in the peritoneal cavity was carried out. METHODS: A suspension of viable adenocarcinoma cells was introduced into the left upper quadrant of the peritoneal cavity of 36 syngeneic immune-competent rats at laparotomy, laparoscopy with CO2 insufflation, and gasless laparoscopy (12 rats in each group). Six days later the peritoneal cavity and surgical wounds were examined for macroscopic evidence of implanted tumour. The abdominal cavity was divided into sectors and macroscopic tumour implantation was determined for each sector and wound. This was confirmed by histological examination. RESULTS: While tumour implantation occurred in the vicinity of the tumour suspension introduction site in the laparotomy and gasless laparoscopy groups, implantation occurred throughout the peritoneal cavity, including areas remote to the introduction site, in the laparoscopy with CO2 insufflation group. Tumour growth was more likely in the port wounds of rats undergoing laparoscopy with insufflation than without. CONCLUSIONS: In this model, CO2 insufflation during laparoscopy resulted in widespread tumour dissemination and implantation, when compared to laparotomy and gasless laparoscopy, supporting the postulate that wound metastasis and tumour spread may be more likely following laparoscopic cancer surgery in humans when CO2 insufflation is used.

Adenocarcinoma↗

PubMed1997

(D)-penicillamine increases hepatic oxalate production resulting in hyperoxaluria.

PURPOSE: To determine whether (D)-penicillamine is effective in reducing hepatic oxalate production and urinary oxalate excretion. MATERIALS AND METHODS: (D)-Penicillamine was administered orally to rats to determine its effect on urinary oxalate excretion and used in isolated rat hepatocytes to investigate the effect of (D)-penicillamine on oxalate production from glycolate. Studies involving hepatic aminotransferases and hepatocytes isolated from (D)-penicillamine treated rats were used to clarify the discrepancy between the in vitro and in vivo results. RESULTS: In hepatocytes (D)-penicillamine lead to a significant reduction in oxalate production from glycolate. In vivo however. (D)-penicillamine led to a significant increase in urinary oxalate excretion and a decrease in plasma aminotransferase activity. Hepatic aminotransferases are involved in diverting oxalate precursors from oxalate production. In vitro, (D)-penicillamine was shown to inhibit hepatic aminotransferases. Hepatocytes isolated from (D)-penicillamine-treated rats produced significantly more oxalate than controls. CONCLUSIONS: These results indicate that (D)-penicillamine increases hepatic oxalate production and urinary oxalate excretion. (D)-penicillamine therefore has no therapeutic potential for reducing endogenous oxalate production and urinary oxalate excretion. Moreover, in conditions such as Wilson's Disease which is often associated with hypercalcuria, its use may be contraindicated.

Animals↗

PubMed1996

Trace metal, acute phase and metabolic response to endotoxin in metallothionein-null mice.

Accumulation of hepatic zinc via metallothionein (MT) induction during infection/inflammation is postulated to benefit a range of metabolic processes. The metabolic consequences of two doses of endotoxin (LPS) (1 and 5 mg/kg, intraperitoneally) were examined in normal (MT+/+) and MT-null (MT-/-) mice (all results means =/- S.E.M., n=6). At 16 h after 1 mg/kg LPS, hypozincaemia was pronounced in the MT+/+ mice (4.4+/-0.2 microM), concomitant with a 36% increase in hepatic Zn and a > 10-fold increase in hepatic MT. Plasma Zn (16.6+/-0.7 microM) and total hepatic Zn were unchanged in MT -/- mice, confirming the importance of MT in altering plasma and hepatic Zn during inflammation. Plasma iron was lower in LPS-treated MT-/- mice, whereas plasma copper increased to a similar extent in both groups of mice. Plasma fibrinogen more than doubled, and was similar in both groups of mice, which questions the importance of MT in acute-phase protein synthesis. Blood and liver glucose concentrations were not significantly different between groups before or after LPS, whereas blood and liver lactate concentrations were significantly lower (31% and 24% respectively) in MT-/- mice after LPS. At 16 h after 5 mg/kg LPS, plasma Zn was decreased even further in MT+/+ mice (2.6+/-0.3 microM), but remained unchanged in MT-/- mice at concentrations significantly above those in 16-h fasted MT-/- mice (15.8+/-0.5 versus 11.3+/-0.3 microM). Total liver Zn was 17% lower than fasting values in MT-/- mice, in contrast with 32% higher in MT+/+ mice. Synthesis of MT (in MT+/+ mice) and fibrinogen in all mice was not further enhanced by the higher LPS dose. Blood glucose was significantly decreased by 18% in MT+/+ mice and by 38% in MT-/- mice after 5 mg/kg LPS. There was a marked 44% decrease in liver glucose in MT-/- mice; that in MT+/+ mice was unchanged from fasting levels, implying a deficit in hepatic gluconeogenesis in LPS-treated MT-/- mice. In the absence of any indication of major hepatotoxicity, the results of this study indicate that energy production, and not acute-phase protein synthesis, may be most influenced by Zn supply during endotoxaemia, suggesting that MT has a role in maintaining hepatic and blood glucose in this metabolic setting.

Animals↗

PubMed1996

Wound metastases following laparoscopic and open surgery for abdominal cancer in a rat model.

The recent application of laparoscopic resection techniques to malignant disease has raised safety concerns due to metastasis to surgical access wounds. The significance and incidence of this problem are controversial. In the present study a rat model, in which an implanted tumour was lacerated, was used to investigate whether application of laparoscopic techniques for malignant abdominal disease leads to an increased risk of tumour dissemination and implantation within the peritoneal cavity, and abdominal wall wounds. Malignant cells were implanted into the abdominal wall of 42 rats, resulting 7 days later in the growth of a tumour measuring 20-25 mm in diameter. There were three control groups: no surgery (n = 6); blunt manipulation of the tumour laparoscopically (n = 6); and blunt manipulation of the tumour at laparotomy (n = 6). Twenty-four rats underwent surgical laceration of the tumour capsule at either laparoscopy (n = 12) or laparotomy (n = 12). All rats were killed 1 week later, and examined for macroscopic evidence of tumour metastasis. The abdominal surgical wounds were excised for independent microscopic examination by a histopathologist. Growth of the primary tumour was greater in rats that had an operation than in unoperated controls, and was greater after laparotomy. However, wound metastases were five times more likely after laparoscopic tumour laceration than after the same procedure through an open incision (ten of 12 rats versus two of 12, P = 0.0033). Wound metastases following laparoscopic tumour manipulation are an important and real problem, with significant implications for the application of laparoscopic techniques to excise malignant disease in humans.

Abdominal Muscles↗

PubMed1996

In vivo studies of macrophages and intercellular adhesion molecule-1 following lipopolysaccharide treatment in tumour-bearing rats.

Histological, haematological and immunocytochemical analyses were performed on a mammary adenocarcinoma implanted in Dark Agouti rats treated with LPS to further elucidate the role of TNF-alpha in tumour biology. Tumours were examined 4-12 days after implantation with parallel studies on rats treated with LPS (200 micrograms/100 g bodyweight [b.w.]) 24 h before removal. Tumour tissue from rats with 12-day-old lesions was also examined over the 24 h period after LPS administration. Haemorrhagic necrosis was evident 6 h after endotoxin treatment and became profound by 24 hours, at which stage only a thin band of viable cells was seen at the rim of the tumour. Using immunocytochemical techniques and a monoclonal anti-macrophage antibody, macrophage numbers were seen to increase throughout the tumour following LPS administration at each stage of tumour growth. Immunocytochemical staining for intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule normally involved in monocyte recruitment, could not be detected in vascular channels within the tumours investigated. However, subcutaneous vessels at the tumour periphery produced a strong linear reaction. The results show that macrophage numbers in tumours increase after LPS administration, concurrently with the development of haemorrhagic necrosis. This cellular infiltration was associated with peripheral monocytopenia. ICAM-1 expression on the vascular endothelium within tumours was deficient.

Adenocarcinoma↗

PubMed1996

Idiopathic calcium oxalate urolithiasis and endogenous oxalate production.

Despite the great effort that has gone into investigating urolithiasis, this condition still persists as one of the major ailments of the urinary tract. Calcium oxalate urolithiasis is the most common form, accounting for some 60 to 80% of total stones. This review examines the elements (i.e., urine volume and pH and urinary excretion of calcium, oxalate, citrate, urate, magnesium, pyrophosphate, and glycosaminoglycans) that give rise to idiopathic calcium oxalate urolithiasis. Treatment strategies for idiopathic calcium oxalate urolithiasis, including lithotripsy, also are discussed. Urinary oxalate excretion is a major risk factor for calcium oxalate urolithiasis, with 85 to 95% of the urinary load derived endogenously. The factors controlling endogenous oxalate production are reviewed, including pathways for the diversion of glyoxylate from oxalate production. The use of beta-aminothiols and other substances to reduce endogenous oxalate production in subjects with idiopathic calcium oxalate urolithiasis is also discussed. A review of current methodologies for the determination of urinary oxalate is also included.

Animals↗

PubMed1995

Hepatic zinc in metallothionein-null mice following zinc challenge: in vivo and in vitro studies.

Hepatic zinc uptake and accumulation were compared in freshly isolated and cultured hepatocytes prepared from control (MT+/+) and metallothionein (MT)-null (MT-/-) mice. In freshly isolated hepatocytes, rapid (10-15 min) exchange of 65Zn was proportional to the Zn concentration in the medium and occurred to the same extent in hepatocytes from MT+/+ and MT-/- mice. In 24 h culture experiments with MT+/+ and MT-/- hepatocytes it was shown that approx. 40% of newly acquired cell-associated Zn was attached to the cell surface and not internalized. In MT+/+ and MT-/- hepatocyte cultures, internalized Zn (intZn) increased in proportion to extracellular Zn. Zn accumulation in MT-/- hepatocytes was only 60% that of MT+/+ cells. Addition of 1 microM dexamethasone (Dex) and recombinant mouse interleukin-6 (IL-6; 100 units/ml) increased MT accumulation by 8.6-fold in MT+/+ hepatocytes (at 50 microM Zn) and there was an associated parallel increase in intZn. Dex and IL-6 did not increase intZn in the MT-/- hepatocytes. At 16 h after an intraperitoneal injection of 5 micrograms/g Zn, plasma and urine Zn concentrations were 69 +/- 10 microM and 86 +/- 25 microM respectively in MT-/- mice (n = 10) and 27 +/- 1 microM and 23 +/- 4 microM respectively in MT+/+ controls (n = 9) (P < 0.001, plasma; P < 0.05, urine). Hepatic cytosolic Zn concentrations doubled in MT+/+ mice and increased by a significant 15% in MT-/- mice. There was no increase in hepatic Zn (dry wt.) concentrations or in total hepatic Zn, demonstrating that the increase in cytosolic Zn in MT-/- mice was due to hepatic water loss rather than net Zn uptake. It appears that even at extreme plasma concentrations of Zn, little if any accumulates within the liver when there is no MT available for its sequestration. That this is not fully demonstrated in vitro is probably due to nature of cell culture, where organ architecture is lost and the external protein binding milieu is less complex.

Animals↗

PubMed1995

Endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression.

The action of endotoxin lipopolysaccharide (LPS) on hepatic Zn uptake was examined in mice lacking expression of metallothionein (MT)-1 and MT-II genes. Hepatic Zn concentrations, which in normal control mice increased by a mean 29% (MT elevated 20-fold) 16 h post-LPS exposure, did not increase in MT-null mice. Plasma Zn fell by 68% in controls and 32% in MT-null mice. The time course of LPS action in normal mice was characterized by a rapid reduction (-74% at 4 h, -81% at 8 h) and partial recovery (-39% at 24 h) in plasma Zn, with a progressive increase over 24 h in hepatic concentrations of MT (by 36-fold) and Zn (by 40%). In contrast, the MT-null mice had a linear decrease in plasma Zn (-15% at 8 h, -41% at 24 h) and early loss of Zn from the liver. The Zn changes seen in MT-null mice were largely attributable to LPS-associated anorexia. Food deprivation (20 h) alone caused respective 14% and 30% decreases in hepatic and plasma Zn concentrations and a 27% reduction in total liver Zn reserves, whereas fasted normal mice conserved Zn with a 4-fold increase in hepatic MT. This study confirms that MT synthesis is essential for endotoxin-induced liver Zn accumulation.

Animals↗

PubMed1995

Metallothionein induction in cultured rat hepatocytes by arthritic rat serum, activated macrophages, interleukin-6, interleukin-11 and leukaemia inhibitory factor.

Potential mediators of hepatic metallothionein (MT) synthesis in adjuvant-induced arthritis were investigated in cultured rat hepatocytes. Sera from arthritic rats (14 d post-adjuvant treatment) in the presence of Zn (50 mumol/L)+dexamethasone (Dex; 1 mumol/L) increased metallothionein (MT) accumulation by 34% above that obtained with control rat serum with Zn+Dex. Endogenous IL-6 activity in serum from arthritic rats was 93 +/- 49 U/mL and was undetectable in control rat serum. The activities of TNF, IL-1 and corticosterone concentrations were the same in control and arthritic rats. The accumulation of MT in hepatocytes in the presence of Zn (10 mumol/L)+Dex (1 mumol/L) was enhanced 29% and 49% by media from lipopolysaccharide (LPS)-stimulated peritoneal macrophage (PMM) and Kupffer cell cultures (KCM), respectively. The response with PMM and KCM was quantitatively the same as that with interleukin-6 (IL-6). Analysis of PMM and KCM showed activities of 1,000-10,000 U/mL for IL-6, 100-1000 U/mL for TNF and < 10,000 U/mL for IL-1, the latter detected only in PMM. LPS alone enhanced the accumulation of MT above Zn+Dex in a dose dependent manner. A significant LPS response was obtained at 5 mg/L with a maximal stimulation above Zn+Dex of 38% at 10 mg/L. This direct stimulation of MT by LPS was not part of the response observed with PMM and KCM where the final LPS concentration in culture was only 0.1 mg/L. Other cytokines capable of synergy with Zn+Dex on MT synthesis were investigated. Interleukin-11 (IL-11) increased the Zn+Dex induction in a dose dependent manner with maximal stimulation at 100 U/mL of 40%. A small stimulation of 12% above Zn+Dex was obtained with leukaemia inhibitory factor (LIF) at concentrations greater than 100 U/mL. No enhancement of the Zn+Dex response was obtained with interleukin-3 (1000 U/mL), interleukin-4 (10 micrograms/L), platelet activating factor (5 nmol/L) or granulocyte-colony stimulating factor (5 micrograms/L). Neither IL-11 nor LIF enhanced the response obtained with Zn+Dex+IL-6. The results demonstrate that mediators present in arthritic rat serum and in LPS-stimulated PMM and KCM cause a quantitatively similar response on MT accumulation as IL-6. IL-11 and to a lesser extent LIF, are also potential mediators of MT synthesis in inflammation.

Animals↗

PubMed1994

Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship to metallothionein and intracellular zinc.

Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetate], a new intracellular zinc fluorophore, was used to reveal and to measure Zn in cultured rat hepatocytes before and after metallothionein (MT) induction. Hepatocytes labelled with an intense extranuclear fluorescence. Culture with combinations of Zn, dexamethasone and interleukin-6, increased intracellular MT by 24-fold, Zn 3-fold, and Zinquin fluorescence by approx. 2-fold above control values. Zinquin fluorescence correlated in descending order with the total cellular Zn (r = 0.747), exchangeable Zn (r = 0.735), soluble cytosolic Zn (r = 0.669) and MT (r = 0.666). When Zinquin was incubated with a cytosolic fraction of liver proteins before Sephadex G-75 column chromatography, it fluoresced with free, MT-incorporated and protein-bound Zn. Although only a slight attenuation of fluorescence was seen with high-molecular-mass protein-bound Zn, MT was degraded by 60% in the presence of Zinquin. The undegraded Zn-MT fluoresced at about 20% of the expected intensity. Although Zinquin fluoresces with all cytosolic Zn, caution is required when comparisons are made between samples with different concentrations of MT. This limitation was demonstrated by staining liver slices from adjuvant-treated rats where MT was increased 24-fold, intracellular Zn by 77%, but Zinquin fluorescence by only 19% above controls. Nevertheless, Zinquin should prove to be a useful tool for studying the distribution of Zn in living cells.

Animals↗