Metallothionein in human oesophagus, Barrett's epithelium and adenocarcinoma.

The potential of the metal-binding protein, metallothionein, in assessing the progression of normal oesophagus through Barrett's to adenocarcinoma was investigated. Metallothionein was quantitatively determined in resected tissues from patients undergoing oesophagectomy for high grade dysplasia/adenocarcinoma and in biopsies from patients with Barrett's syndrome. In 10 cancer patients, metallothionein concentrations in adenocarcinoma were not significantly different from normal oesophagus, although six had elevated metallothionein concentrations in the metaplastic tissue bordering the adenocarcinoma. In 17 out of 20 non-cancer patients with Barrett's epithelium, metallothionein was significantly increased by 108% (P<0.004). There was no association between the metallothionein levels in Barrett's epithelium and the presence of inflammatory cells, metaplasia or dysplasia. Metallothionein is a marker of progression from normal to Barrett's epithelium but is not increased in oesophageal adenocarcinoma.

Metallothionein: the multipurpose protein.

Metallothioneins (MTs) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTs have unique structural characteristics to give potent metal-binding and redox capabilities. A primary role has not been identified, and remains elusive, as further functions continue to be discovered. The most widely expressed isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators. In teh gut and pancreas, MT responds mainly to Zn status. A brain isoform, MT-3, has a specific neuronal growth inhibitory activity, while MT-1 and MT-2 have more diverse functions related to their thiolate cluster structure. These include involvement in Zn homeostasis, protection against heavy metal (especially Cd) and oxidant damage, and metabolic regulation via Zn donation, sequestration and/or redox control. Use of mice with altered gene expression has enhance our understanding of the multifaceted role of MT, emphasised in this review.

Importance of storage conditions for the stability of zinc- and cadmium-induced metallothionein.

We examined the storage stability of metallothionein (MT), a cysteine-rich protein that has diagnostic potential as a cancer marker and in the assessment of Zn status and heavy-metal toxicity. MT was rapidly degraded in samples of rat whole liver at -20 degrees C or -70 degrees C. MT in supernatants from heat-treated rat liver homogenates stored as 1:5 dilutions of liver from Zn- or Cd-induced rats were stable (recovery >98%) for 100 d at temperatures of -70 degrees C and -196 degrees C but not at -20 degrees C, regardless of the presence of dithiothreitol (DTT) or argon. The variability of MT measurement by the 109Cd-hemoglobin affinity assay was however greatest in samples from Zn-induced rats stored without DTT. The integrity of the MT protein in supernatants of heat-treated homogenates stored for 100 d was demonstrated by Sephadex G-75 chromatography. When heat-treated supernatants were stored as dilute solutions (1:125 of liver), MT was unstable regardless of treatment or storage temperature. Our findings show that liver MT is stable for at least 4 mo as a supernatant of a heat-treated homogenate (1:5 dilution of liver) when stored at or below -70 degrees C and in the presence of DTT.

Influence of gases on intraperitoneal immunity during laparoscopy in tumor-bearing rats.

Laparoscopy has been associated with metastases to abdominal wall wounds. In addition, many recent experimental studies suggest that laparoscopy is associated with increased tumor dissemination. It is possible that immune or metabolic disturbances due to the use of a pneumoperitoneum could contribute to this problem. To investigate this possibility, we studied the effect of two insufflation gases and gasless laparoscopy on in vivo peritoneal macrophage function and intraperitoneal pH in an experimental model. A carcinoma was implanted into the flank of 32 experimental rats that underwent laparoscopic surgery in one of four treatment groups: anesthesia alone, gasless laparoscopy, helium insufflation, and CO2 insufflation. Intraperitoneal pH was monitored during surgery, and peritoneal macrophage function was determined 3 days after surgery by harvesting peritoneal macrophages and then examining their ability to produce tumour necrosis factor-alpha (TNF-alpha). CO2 insufflation was associated with a consistent fall in intraperitoneal pH and a significant reduction in TNFalpha production. These findings did not occur in the other study groups. The results of this study demonstrate that CO2 insufflation results in depressed intraperitoneal macrophage activity. It is possible that it is mediated by pH changes. In addition, it could be a contributing factor to the development of port-site metastases. Further studies are needed to determine whether the factors identified act during clinical surgery.

Activation of glycolysis by zinc is diminished in hepatocytes from metallothionein-null mice.

The influence of hepatic metallothionein (MT) and zinc (Zn) on glycolysis was investigated in primary cultures of mouse hepatocytes prepared from MT-normal (+/+) and MT-null (-/-) mice. In MT +/+ mice, a close relationship was observed between the Zn concentration in the incubation medium (10-150 microM), increased MT levels in the cells, and increased glycolysis (accumulation of lactate + pyruvate) over 24 h, with significant effects seen at physiological levels of Zn (10-25 microM). Hepatocytes from MT -/- mice had significantly lower basal rates of glycolysis and demonstrated increased glycolysis only at Zn concentrations of 50 microM or greater. The lactate:pyruvate ratio was higher in the MT +/+ hepatocytes. The oxidation of endogenous fatty acid (accumulation of the ketone bodies, 3-hydroxybutyrate and acetoacetate) was initially greater in the MT +/+ hepatocytes, although only MT -/- hepatocytes showed increased ketone body production in response to Zn. The 3-hydroxybutyrate:acetoacetate ratio was higher in the MT +/+ hepatocytes and increased with increasing Zn concentrations. Intracellular Zn accumulation was 60% greater in the MT +/+ hepatocytes, with approximately 80% of the extra Zn associated with MT. The results implicate MT-associated Zn rather than increased intracellular Zn per se in the regulation of hepatic carbohydrate metabolism.

Ethanol decreases zinc transfer to the fetus in normal but not metallothionein-null mice.

BACKGROUND: Ethanol causes significant teratogenicity in normal (MT+/+) but not metallothionein-null (MT-/-) fetuses. Impaired maternal fetal zinc (Zn) transfer is indicated, because ethanol significantly reduces plasma Zn concentrations in MT+/+ dams while increasing concentrations in MT-/- dams. In this study we examined maternal-fetal Zn homeostasis in response to ethanol in MT+/+ and MT-/- mice and the origins of the increase in plasma Zn in MT-/- mice. METHODS AND RESULTS: Mice were treated with saline or ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) on day 12 of gestation. An additional subcutaneous injection of 65Zn tracer was administered after the second ethanol injection before mice were killed 3 hr later. Maternal liver MT levels were not different between ethanol and saline MT+/+ mice. Both liver Zn and 65Zn levels were higher in MT+/+ mice. Plasma Zn concentrations were higher in MT-/- mice, with MT-/- ethanol-treated mice having levels greater than those of MT-/- saline-treated controls. MT+/+ ethanol-treated fetuses exhibited lower 65Zn transfer and whole Zn concentrations compared with MT+/+ and MT-/- saline and MT-/- ethanol fetuses. So we could examine changes in plasma Zn after ethanol treatment, MT+/+ and MT-/- mice were injected with 65Zn 3 days before they received ethanol treatment. Muscle and skin showed a decrease in 65Zn retention in both genotypes over 3 hr. There was a trend toward greater 65Zn release from skin and muscle at an earlier time in MT-/- mice: 24% vs. 2% decrease (MT-/- vs. MT+/+) for muscle and 28% vs. 15% decrease (MT-/- vs. MT+/+) for skin at 2 hr. CONCLUSIONS: The results show (a) that ethanol interferes with the transfer of Zn to the fetus, and that this is MT dependent, and (b) that the increase in plasma Zn seen in MT-/- mice after ethanol administration is a result of Zn release from the skin and muscle, in the absence of hepatic Zn sequestration.

Tolerance to cadmium hepatotoxicity by metallothionein and zinc: in vivo and in vitro studies with MT-null mice.

The protective role of metallothionein (MT) in Cd-mediated hepatotoxicity was investigated in vivo and in vitro. Following injection of Cd (2 mg/kg, intraperitoneal or subcutaneous) hepatoxicity was significantly greater at 20 h in metallothionein-null (MT-/-) mice, compared with controls (MT+/+). The decrease in the blood and liver glucose concentrations correlated with the extent of hepatotoxicity, with blood glucose 43% lower in MT-/- mice. Zinc (50 microM) and/or Dex (1 microM) were used in hepatocyte cultures to raise MT 2-5-fold. When Cd at 10 microM was co-treated with Zn and/or Dex, lactate dehydrogenase (LD) leakage in the MT+/+ and MT-/- hepatocytes was reduced only when Zn was present. Cellular glutathione (GSH) was the same in control MT+/+ and MT-/- cultures and was uninfluenced by Zn and Dex. After treatment with 5 and 10 microM Cd, GSH levels were lower in MT-/- than MT+/+ hepatocytes in the control and Dex groups. Higher GSH concentrations were maintained in Zn co-treated cultures from both genotypes, indicating that the superior protective effect of Zn may in part derive from its influence on cellular GSH. Pre-treatment with Zn and/or Dex provided no further protection than co-treatment. Tolerance to brief (15 min) Cd exposure was also investigated in the presence of MT inducers including progesterone (100 microM). Zn, Dex and progesterone treated hepatocytes had less LD leakage than controls with Zn giving the greatest protection (LD leakage 18% of controls at 100 microM Cd). Zn pre-treated cells had higher cytosolic/particulate ratios of Cd. These findings demonstrate that MT protects primary cultures of mouse hepatocytes from short-term exposure to Cd. Zn enhances the protection through MT and non-MT mechanisms.

Metallothionein in mice reduces intestinal zinc loss during acute endotoxin inflammation, but not during starvation or dietary zinc restriction.

Normal metallothionein [(MT)+/+] and MT-null (MT-/-) mice were used to examine the influence of MT on Zn retention and the metabolic consequences of 2 d food deprivation, with and without inflammation induced by intraperitoneal injection of bacterial endotoxin lipopolysaccharide (LPS). LPS reduced fecal Zn concentration in MT+/+ mice from 5.9 +/- 0.2 micromol/g on d 1 to 2.2 +/- 0.2 micromol/g on d 2, but not in MT-/- mice, 5.9 +/- 0.2 and 5.7 +/- 0. 5 micromol/g, respectively. MT+/+ mice fed an 8 mg Zn/kg diet and injected with LPS excreted 40% less Zn over 2 d than their MT-/- counterparts. Starvation for 2 d did not lower fecal Zn concentration in either genotype, although in MT+/+ mice, urinary Zn excretion was reduced from 12.7 +/- 1.3 nmol on d 1 to 5.9 +/- 1.8 nmol on d 2 and plasma Zn concentration was lowered to 9.8 +/- 0.4 micromol/L. Zn was not reduced in urine or plasma of MT-/- mice, with respective values of 10.8 +/- 2.0 nmol on d 1, 9.3 +/- 2.9 nmol on d 2 and 13.0 +/- 1.0 micromol/L. LPS injection resulted in much higher total liver Zn (677 +/- 27 nmol) and MT (106 +/- 2 nmol Cd bound/g) than starvation (Zn = 405 +/- 21, MT = 9 +/- 3) in MT+/+ mice after 2 d, but did not further reduce urinary Zn. LPS-injected MT-/- mice had no rise in liver Zn or fall in plasma and urine Zn. MT-/- mice fed a Zn-deficient (0.8 mg Zn/kg) diet lost 10% of body weight over 25 d compared with no loss in MT+/+ mice. Despite this, MT-/- mice excreted no more Zn via the gut than did MT+/+ mice. In summary, MT inhibits intestinal Zn loss when highly expressed. When uninduced, typically during Zn deficiency, MT appears to conserve Zn and body mass by reducing only urinary and other nonintestinal Zn losses.

The effect of immune enhancement and suppression on the development of laparoscopic port site metastases.

BACKGROUND: Recent clinical case reports and experimental studies have suggested that laparoscopic cancer surgery is associated with an increased risk of tumor spread to abdominal wall wounds. While the etiology of this problem was initially believed to be related to mechanical contamination of wounds, it is now recognized that there are other contributory factors, including disturbed immune function within the peritoneal cavity. To investigate this question further, we evaluated the effect of immune modulation within an established laparoscopic cancer model. METHODS: Eighteen immune-competent syngeneic rats underwent modulation of their immune system, followed 18 h later by laparoscopy with the introduction of a suspension of adenocarcinoma cells into the peritoneal cavity. Rats were randomly allocated to receive either systemic cyclosporin (immune suppressor), intraperitoneal endotoxin (immune enhancer), or no agent (controls). Seven days later, all rats were killed and their peritoneal cavity was inspected for tumor implantation and port site metastases. RESULTS: Cyclosporin did not influence the study outcome, but tumor growth (p = 0.008) and port site metastases (p < 0.0001) were less common following the administration of intraperitoneal endotoxin. CONCLUSION: The results of this study suggest that the immune system plays a role in the genesis of port site metastases. A preventive role for endotoxin in patients undergoing laparoscopic cancer surgery, however, remains speculative.

Zn-depleted mice absorb more of an intragastric Zn solution by a metallothionein-enhanced process than do Zn-replete mice.

The influence of metallothionein (MT)(2) on Zn absorption was investigated in MT-null (MT-/-) and normal (MT+/+) mice fed Zn-depleted (ZnD) diets for 7 d and compared with those fed Zn-replete (ZnR) diets in a previous study. Mice were starved for 20 h, then administered an oral gavage of aqueous (65)ZnSO(4) solution at doses of 154, 770 or 1540 nmol of Zn, and the amount transferred into nongut tissues was determined 4 h later. (65)Zn transfer did not differ between genotypes in ZnR mice. However ZnD MT+/+ mice had a 30-40% greater transfer from the 154 and 770 Zn doses compared to ZnR MT+/+ mice. This was not observed in MT-/- mice. In MT+/+ mice, Zn depletion enhanced the induction of MT by Zn in the intestine and pancreas. (65)Zn uptakes in the liver and pancreas were greater in MT+/+ than MT-/- mice, and this was greater (50%) at the 154 and 770 doses in mice fed ZnD diets. Plasma Zn concentrations were raised to a similar extent in ZnR and ZnD MT-/- mice. ZnR MT+/+ mice had significantly lower plasma Zn levels than MT-/-mice; this difference was less marked in the ZnD mice. We conclude that a MT-facilitated enhancement in Zn absorption occurs in response to dietary Zn deficiency.

Maternal ethanol exposure is associated with decreased plasma zinc and increased fetal abnormalities in normal but not metallothionein-null mice.

BACKGROUND: Ethanol profoundly affects fetal development, and this is proposed to be due primarily to a transient fetal zinc (Zn) deficiency that arises from the binding of Zn by metallothionein (MT) in the maternal liver. Zn homeostasis and fetal outcome were investigated in normal (MT+/+) and metallothionein-null (MT-/-) mice in response to ethanol exposure. METHODS/RESULTS: Mice were treated with saline or ethanol (0.015 m/g intraperitoneally at 0 and 4 hr) on day 8 of gestation (Gd8), and the degree of fetal dysmorphology was assessed on Gd18. The incidence of external abnormalities was significantly increased in offspring from MT+/+ dams exposed to ethanol, where 27.4% of fetuses were affected. MT-/- ethanol-, MT+/+ saline-, and MT-/- saline-treated dams had fetuses in which the frequencies of abnormalities were 2.2, 6.4, and 6.9%, respectively. To investigate Zn homeostasis, nonpregnant mice were killed at intervals over 16 hr after ethanol injection. Liver MT concentrations in MT+/+ mice were increased 20-fold by 16 hr, with a significant elevation evident by 4 hr, whereas liver Zn levels were also significantly increased by 2 hr and maintained for 16 hr. In parallel with these changes, plasma Zn concentrations in MT+/+ mice decreased by 65%, with minimum levels of 4.5+/-0.3 micromol/liter at 8 hr. Conversely, MT-/- mice exhibited increased plasma Zn concentrations, with peak values of 20.8+/-0.3 observed at 4 hr. CONCLUSION: These findings link the teratogenic effect of ethanol to the induction of maternal MT and the limitation of fetal Zn supply from the plasma.

Increased zinc absorption but not secretion in the small intestine of metallothionein-null mice.

Metallothionein (MT) has been assigned a role in intestinal Zn absorption and secretion. The influence of MT was investigated in isolated segments of the small intestine from mice lacking the expression of MT I and II genes (MT-/-). To measure Zn absorption, washed 10- to 12-cm segments of the proximal and distal small intestine of MT-/- and control MT+/+ mice were filled with 65Zn as ZnSO4 (10 microg/mL), and the amount of 65Zn appearing in the external buffer was measured over 4 h. To measure Zn secretion, the same procedure was followed using everted gut segments. The 65Zn absorption from the small intestine was significantly greater in MT-/- mice, but only in the absence of albumin. In the proximal small intestine, the inclusion of 2% albumin in the external buffer significantly increased Zn absorption from 6.8% (no albumin) to 13.2% (with albumin) for MT-/-, and from 4.9% (no albumin) to 14.2% (with albumin) for MT+/+. In the distal segment, the respective values, with and without albumin respectively were 9.5% and 15.1% for MT-/- mice and 4.3% and 16.1% for MT+/+ mice. Regarding 65Zn secretion, there was no difference between MT+/+ and MT-/- in either segment. However, the rate of secretion was higher in the proximal small intestine for both genotypes. Although it can be demonstrated that MT limits Zn absorption under controlled conditions in vitro, the ability of albumin to overcome this effect emphasizes the importance of circulating ligands in Zn transport.

Regional distribution and localization of zinc and metallothionein in the intestine of rats fed diets differing in zinc content.

BACKGROUND: Zinc (Zn) is protective and enhances epithelial repair in gut diseases. In this study we investigate the localization and distribution of Zn and its binding protein, metallothionein (MT), in the gut of rats fed diets varying in Zn content. METHODS: Male-Sprague Dawley rats were fed low, normal, high, or excess Zn in their diets (10, 100, 400, or 1000 mg Zn/kg, respectively) and killed 7 days later. Blood, liver, and gut tissues were collected. Tissue Zn was determined with atomic absorption spectrophotometery and MT with a Cd/haem affinity assay. Zn and MT were immunohistochemically localized in the small-intestinal wall with zinquin and an anti-MT antibody. RESULTS: Most Zn in the intestinal wall was present in the mucosal scrapings, with 94% membrane-bound and 6% cytosolic, irrespective of dietary Zn. MT levels increased in all gut regions at dietary Zn levels above 100 mg Zn/kg. MT was 40% higher in the ileum than in other gut regions in rats fed low- and normal-Zn diets. The Zn content of the ileum was also 20% higher than that of other gut regions in rats fed low-, normal-, or high-Zn diets. Zn and MT were colocalized in the base of the intestinal crypts, most visibly in the ileum. CONCLUSION: Mucosal cytosolic Zn and MT concentrations are increased only at high or excessive Zn intakes in all gut regions except the ileum, which can respond to a lower Zn intake. As the cytosolic Zn pool most likely influences mucosal protection and repair mechanisms, it is proposed that an increased MT may indicate the adequacy of oral Zn therapy in gut disease.

The role of the pancreas in intestinal zinc secretion in metallothionein-null mice.

The distribution and excretion of endogenous Zn, including the role of the pancreas, were examined in fasted MT+/+ and MT-/- mice. At 3 and 6 h after receiving 65Zn tracer by subcutaneous injection, 65Zn levels were compared in tissues of MT+/+ and MT-/- mice. 65Zn levels were significantly higher in the liver and pancreas of the MT+/+ mice, whereas in the MT-/- mice, 65Zn levels were significantly higher in muscle, skin, and most of the gastrointestinal tract other than the stomach and upper small intestine. In MT-/- mice, 3% of the injected 65Zn was recovered in the luminal contents of the small intestine over 3-6 h, compared with <1.5% in the MT+/+ mice. A loading dose of Zn (150 microg, s.c.) sufficient to raise the plasma Zn concentration by fourfold to fivefold in both MT+/+ and MT-/- mice resulted in similar increases in pancreatic Zn levels in each genotype, although more Zn appeared in the lower small intestine of MT-/- mice. Pancreatectomy decreased the level of 65Zn in the small intestine of MT-/- but not MT+/+ mice. Longer-term studies over 4 days demonstrated few differences in tissue 65Zn between MT+/+ and MT-/- mice, with the exception of the pancreas, where 65Zn retention after fasting in MT-/- mice was half that of MT+/+ mice. MT-/- mice also had significantly lower Zn concentrations in the pancreas. Fecal excretion of 65Zn in MT-/- mice was greater than that of MT+/+ mice in the first 24 h (24.7 vs. 18.2% of injected dose; p < 0.05). Besides metallothionein (MT), there were no significant differences in the molecular weight distribution of Zn binding ligands in the lumen of the small intestine between MT+/+ and MT-/- mice. Mice lacking MT I and II lose more endogenous Zn into the gut because of a relative failure of the pancreas to retain Zn. However, increased Zn secretion via the small intestinal mucosa may also contribute to intestinal Zn loss in MT-/- mice.

Influence of cytotoxic agents on intraperitoneal tumor implantation after laparoscopy.

PURPOSE: Recent experimental studies suggest that laparoscopic surgery for abdominal malignancy may be associated with increased tumor implantation. This study investigated the influence of cytotoxic agents (administered intraperitoneally or intramuscularly) on implantation of a tumor cell suspension after laparoscopic surgery in an experimental model. METHODS: Thirty-three Dark Agouti rats underwent laparoscopy with CO2 insufflation and instillation of a tumor cell suspension into the abdominal cavity. Rats were randomly allocated to one of the following study groups (9 rats in the control group, 6 rats in all other groups): 1) control (no intraperitoneal instillation); 2) intraperitoneal normal saline (0.9 percent); 3) intraperitoneal povidone-iodine (Betadine to normal saline 1:10 dilution); 4) intraperitoneal methotrexate (2 doses of 0.125 mg/kg body weight in normal saline administered 24 hours apart); 5) intramuscular injection of 2 doses of 0.125 mg/kg body weight administered 24 hours apart (no intraperitoneal agent). Rats were killed 7 days after the procedure, and the peritoneal cavity and port sites were examined for the presence of tumor. RESULTS: A significant reduction in tumor implantation and port-site metastases was observed in all treatment groups (povidone-iodine and intramuscular and intraperitoneal methotrexate). CONCLUSIONS: This study suggests that tumor implantation after laparoscopic surgery and port-site metastases might be prevented by the intraperitoneal or systemic administration of cytotoxic agents. Further studies are needed to determine whether these findings can be applied to clinical practice.

Metallothionein-null mice absorb less Zn from an egg-white diet, but a similar amount from solutions, although with altered intertissue Zn distribution.

The influence of metallothionein (MT) on Zn transfer into non-gut tissues was investigated in MT-null (MT-/-) and normal (MT+/+) mice 4 h after oral gavage of aqueous 65ZnSO4solution at doses of 154, 385, 770 and 1540 nmol Zn per mouse. Zn transfer was not significantly different between MT+/+ and MT-/- mice and was directly proportional to the oral dose (slope = 0.127, r = 0.991; 0. 146, r = 0.994, respectively). Blood 65Zn and plasma Zn concentrations increased progressively in MT-/- mice at doses >154 nmol Zn, reaching levels of 2.4% of oral dose and 60 micromol/L, respectively, at the 1540 nmol Zn dose. The corresponding values for MT+/+ mice were approximately half, 1.0% and 29 micromol/L. Intergenotypic differences were found in tissue distribution of 65Zn within the body; MT-/- mice had higher 65Zn levels in muscle, skin, heart and brain, whereas MT+/+ mice retained progressively more Zn in the liver, in conjunction with a linear increase in hepatic MT up to the highest Zn dose. MT induction in the small intestine reached its maximum at an oral dose of 385 nmol Zn and did not differ at higher doses. Absorption of a 770 nmol 65Zn dose from a solid egg-white diet was only one fourth (MT+/+) and one eighth (MT-/-) of the Zn absorption from the same dose of 65Zn in aqueous solution. MT+/+ mice had greater (P < 0.05) Zn absorption from the egg-white diet than did MT-/- mice, indicating that gut MT confers an absorptive advantage, but only when Zn is incorporated into solid food.

The role of peritoneal immunity and the tumour-bearing state on the development of wound and peritoneal metastases after laparoscopy.

BACKGROUND: The effect of the tumour-bearing state and alterations in peritoneal immune function on the incidence of port-site and peritoneal metastases was investigated after laparoscopy with and without CO2 pneumoperitoneum. METHODS: A suspension of viable adenocarcinoma cells was introduced into the left upper quadrant of the peritoneal cavity of syngeneic tumour-bearing rats at laparotomy, laparoscopy with CO2, and gasless laparoscopy. Control rats did not have pre-existing tumours. A group of non-tumour-bearing rats were also injected intraperitoneally with endotoxin 4 h before intraperitoneal tumour cell injection. Six days later the peritoneal cavity and surgical wounds were examined for macroscopic evidence of implanted tumour. Peritoneal macrophages were obtained from tumour-bearing rats subjected to different laparoscopic procedures and the activation state measured following exposure to lipopolysaccharide in vitro. RESULTS: In the control rats, tumour implantation in the surgical wounds and peritoneum was significantly greater in the rats that had undergone laparoscopy with CO2. The presence of a pre-existing tumour was associated with increased tumour spread in all treatment groups and at most sites. Injection of endotoxin also resulted in increased tumour spread. Peritoneal macrophages from control and tumour-bearing rats who underwent laparoscopy with CO2 produced significantly less TNF-alpha in vitro, compared to gasless laparoscopy or laparotomy. CONCLUSIONS: Carbon dioxide insufflation enhances tumour spread and implantation. The underlying immune or metabolic status of the host, as influenced by the tumour-bearing state or modification of the peritoneal environment, also has a marked independent effect on tumour spread and implantation. The immune and metabolic status of the peritoneum including the extent of macrophage activation is implicated in this effect.

In vitro inhibition of tumour growth in a helium-rich environment: implications for laparoscopic surgery.

BACKGROUND: The recent results of several experimental studies have suggested that tumour implantation after laparoscopic surgery for intra-abdominal malignancy may be partly related to the chemical composition of the insufflation gas used during surgery. These studies have demonstrated that the use of helium as a laparoscopic insufflation agent for cancer surgery results in less tumour implantation and growth at port sites. To further investigate these findings, the present study was performed to compare the growth of cultured tumour cells after exposure to simulated laparoscopic environments, rich in helium, carbon dioxide (CO2), or air. METHODS: A rat mammary adenocarcinoma cell suspension was exposed to a simulated laparoscopic environment for 40 min in one of the following groups: (i) control (atmospheric air, equivalent to a 'gasless' laparoscopic environment); (ii) a CO2-rich environment; and (iii) a helium-rich environment. Cells were then cultured for 18 h and optical density readings were used to assess the number of viable tumour cells at the end of this period. The experiment was performed twice using an identical protocol to ensure consistency in the results. In a further study, pH was continuously measured using an antimony probe during a 40 min insufflation period and for 10 min after insufflation. RESULTS: Cell growth was significantly lower after incubation in the helium-rich environment compared to both the CO2 and control groups (P < 0.001). There was a significant decrease in pH in the CO2 group which was not observed during exposure to either air or helium. CONCLUSIONS: The inhibition of tumour growth in a helium-rich environment demonstrated by this study, and the reduced incidence of port-site metastases seen in other experimental studies, suggests that the clinical use of helium as an insufflation gas may have important advantages over CO2.

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.

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.