The Role of Intracellular Glutathione in Inorganic Mercury-Induced Toxicity in Neuroblastoma Cells

It is well known that antioxidants containing sulfhydryl (−SH) groups are protective against the toxic effects of mercury. The current study was designed to elucidate the mechanism(s) of the cytoprotective effects of glutathione (GSH) and N-acetylcysteine (NAC) against the toxicity of inorganic mercury (HgCl₂) in neuroblastoma cells (N-2A). The obtained results demonstrated the protective effects of these compounds in a dose dependant manner up to 95 and 74% cell viability, respectively as compared to the control of HgCl₂ of 10%. The administration of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, increased the toxicity of HgCl₂ in a dose dependent manner. Moreover, BSO treatment attenuated the levels of the cellular free −SH concentrations at low concentrations (1–100 μM) of HgCl₂. The data also show that cellular thiol concentrations were augmented in the presence of GSH and NAC and these compounds were cytoprotective against HgCl₂ and this is due to up regulating of GSH synthesis. A reduction in intracellular levels of GSH was observed with treatment of HgCl₂. In addition, the ratio of GSH/GSSG increased from 16:1 to 50:1 from 1 to 10 μM concentration of HgCl_2. The ratio of GSH/GSSG then decreased from 4:1 to 0.5:1 with the increase of concentration of HgCl₂ between 100 μM and 1 mM due to the collapse of the N-2A cells. It was of interest to note that the synthesis of GSH was stimulated in cells exposed to low concentration of HgCl₂ when extra GSH is available. These data support the idea that the loss of GSH plays a contributing role to the toxic effects of HgCl₂ and that inorganic mercury adversely affects viability, through altering intracellular −SH concentrations. The data further indicate that the availability of GSH to the cells may not be sufficient to provide protection against mercury toxicity and the de novo synthesis of intracellular GSH is required to prevent the damaging effects of mercury.     

Lipid peroxidation in rats administrated with mercuric chloride

Parenteral administration of mercuric chloride (HgCl₂) to rats enhanced lipid peroxidation in liver, kidney, lung, testis, and serum (but not in heart, spleen, or muscle), as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates and body fluids. After sc injection of HgCl₂ (5 mg/kg body wt), MDA concentrations in liver and kidney became significantly increased by 9 h and reached peak values at 24 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 1, 3, 5 mg Hg/kg as HgCl₂ and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as HgCl₂ and sacrificed after 3, 9, 18, 24, and 48 h. Studies in the authors' laboratory have shown that (1) concentrations of MDA are increased in targets (liver, kidney, lung, and testis) of HgCl₂-treated rats; (2) severity of hepatotoxicity and nephrotoxicity is generally consistent with the elevation of Hg and MDA concentrations, based upon the time-course and dose-effect relationships observed after administration of HgCl₂ to rats; and (3) concentrations of MDA are reduced in target tissues after pretreatment with antioxidants and chelators to HgCl₂-treated rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute HgCl₂ poisoning.     

Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacteriumSynechococcus 6301

Inhibition of electron transport activities in the spheroplasts ofSynechococcus 6301 by HgCl² is dependent on the concentration of mercury ions. The inhibition of whole chain electron transport activity occurs at low concentration of Hg²⁺ (6 ΜM@#@). This inhibition occurs mostly due to interaction of Hg²⁺ on plastocyanin. At an elevated concentration (24 ΜM@#@), mercury induces inhibition chiefly in photosystem II catalyzed electron transport. At this concentration it also alters both the absorption and emission characteristics of the phycocyanin. The photosystem I catalyzed electron transport was inhibited by 50% only at high concentrations (36 ΜM@#@) of HgCl₂. However, electron transport catalyzed by photosystems I and II from reduced duroquinone to methylviologen which involves intersystem electron transport is extremely sensitive to mercury (low concentration 6–9 ΜM) like that of whole chain assay indicating that the observed inhibition in whole chain electron transport at low concentrations is mostly contributed by the damage involving other intersystem electron transport carrier(s) like plastocyanin. Thus mercury ions depending on the concentration affects the electron transport at multiple sites in the spheroplasts ofSynechococcus.     

Assessment of mercury chloride-induced toxicity and the relevance of P2X7 receptor activation in zebrafish larvae

Zebrafish (Danio rerio) has been adopted as a model for behavioral, immunological and toxicological studies. Mercury is a toxic heavy metal released into the environment. There is evidence indicating that heavy metals can modulate ionotropic receptors, including the purinergic receptor P2X7. Therefore, this study evaluated the in vivo effects of acute exposure to mercury chloride (HgCl₂) in zebrafish larvae and to investigate the involvement of P2X7R in mercury-related toxicity. Larvae survival was evaluated for 24 h after exposure to HgCl_2, ATP or A740003. The combination of ATP (1 mM) and HgCl₂ (20 μg/L) decreased survival when compared to ATP 1 mM. The antagonist A740003 (300 and 500 nM) increased the survival time, and reversed the mortality caused by ATP and HgCl₂ in association. Quantitative real time PCR showed a decrease of P2X7R expression in the larvae treated with HgCl₂ (20 μg/L). Evaluating the oxidative stress our results showed decreased CAT (catalase) activity and increased MDA (malondialdehyde) levels. Of note, the combination of ATP with HgCl₂ showed an additive effect. This study provides novel evidence on the possible mechanisms underlying the toxicity induced by mercury, indicating that it is able to modulate P2X7R in zebrafish larvae.     

Antioxidants and metallothionein levels in mercury-treated mice

Acute effects of mercury on mouse blood, kidneys, and liver were evaluated. Mice received a single dose of mercuric chloride (HgCl₂, 4.6 mg/kg, subcutaneously) for three consecutive days. We investigated the possible beneficial effects of antioxidant therapy (N-acetylcysteine (NAC) and diphenyl diselenide (PhSe)₂) compared with the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), an effective chelating agent in HgCl₂ exposure in mice. We also verified whether metallothionein (MT) induction might be involved in a possible mechanism of protection against HgCl₂ poisoning and whether different treatments would modify MT levels and other toxicological parameters. The results demonstrated that HgCl₂ exposure significantly inhibited δ-aminolevulinate dehydratase (δ-ALA-D) activity in liver and only DMPS treatment prevented the inhibitory effect. Mercuric chloride caused an increase in renal non-protein thiol groups (NPSH) and none of the treatments modified renal NPSH levels. Urea concentration was increased after HgCl₂ exposure. NAC plus (PhSe)₂ was partially effective in protecting against the effects of mercury. DMPS and (PhSe)₂ were effective in restoring the increment in urea concentration caused by mercury. Thiobarbituric acid-reactive substances (TBARS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities and ascorbic acid levels were not modified after mercury exposure. Mercuric chloride poisoning caused an increase in hepatic and renal MT levels and antioxidant treatments did not modify this parameter. Our data indicated a lack of therapeutic effect of the antioxidants tested.     

DMPS and N-acetylcysteine induced renal toxicity in mice exposed to mercury

Acute effects of mercuric chloride (HgCl₂) were evaluated on mice. Mice received a single dose of HgCl₂ (4.6 mg/kg, subcutaneously) for three consecutive days. Thirty minutes after the last injection with HgCl₂, mice received one single injection of 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetylcysteine (NAC) or diphenyl diselenide (PhSe)₂. DMPS, NAC and (PhSe)₂ were utilized as therapy against mercury exposure. At 24 h after the last HgCl₂ injection, blood, liver and kidney samples were collected. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K_-⁺ ATPase activities, thiobarbituric acid-reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid concentrations were evaluated. Plasma aspartate (AST) and alanine (ALT) aminotransferase activities, as well as urea and creatinine levels were determined. The group of mice exposed to Hg + (PhSe)₂ presented 100% of lethality. Exposure with HgCl₂ caused a decrease on the body weight gain and treatments did not modify this parameter. δ-ALA-D, AST and ALT activities, TBARS, ascorbic acid levels and NPSH (hepatic and erythrocytic) levels were not changed after HgCl₂ exposure. HgCl₂ caused an increase in renal NPSH content and therapies did not modify these levels. Mice treated with (PhSe)₂, Hg + NAC and Hg + DMPS presented a reduction in plasma NPSH levels. Creatinine and urea levels were increased in mice exposed to Hg + NAC, while Hg + DMPS group presented an increase only in urea level. Na⁺, K_-⁺ ATPase activity was inhibited in mice exposed to Hg + DMPS and Hg + NAC. In conclusion, therapies with (PhSe)₂, DMPS and NAC following mercury exposure must be better studied because the formation of more toxic complexes with mercury, which can mainly damage renal tissue.     

Lipid peroxidation in liver of rats administrated with methyl mercuric chloride

Parenteral administration of methyl mercuric chloride (MMC, CH₃HgCl) to rats enhanced lipid peroxidation in liver of rats, as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates. After sc injection of CH₃HgCl (5 mg/kg body wt), MDA concentration in liver became significantly increased at 24 h and further increased at 48 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 3 or 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 and 48 h. Studies in the authors’ laboratory have shown that (1) mercury is accumulated in liver; (2) concentration of MDA is increased in liver of CH₃HgCl-treated rats; (3) severity of hepatotoxicity is generally proportional to the elevation of MDA concentration, based upon the dose-effect relationships observed after administration of CH₃HgCl to rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute CH₃HgCl poisoning.     

In-vitro radiometry and microscopy for sensitive measurement of toxicity in activated sludge

Summary Inhibitory effects of an anti-microbial agent on a model activated sludge system were examined. Using microbial counts and¹⁴CO₂ evolution from radiolabeled starch and cellulose, inhibitory effects of small concentration increments of HgCl₂ could be monitored as could the rapid recovery of microbial viability after 24 h. Mercury dissipation accounted for part of this recovery but bacterial resistance to mercury is also implicated.     

The Protective Effects of Tea Polyphenols and Schisandrin B on Nephrotoxicity of Mercury

Mercury (Hg) is an occupational and environmental contaminant that is a well-recognized health hazard. To approach the concrete mechanisms of mercury nephrotoxicity and find out a new way to prevent it, the rats were subcutaneously injected with different dosages of mercuric chloride (HgCl₂)—0, 2.2, 4.4, and 8.8 μmol/kg. The levels of Hg, blood urea nitrogen (BUN), urine protein, glutathione (GSH), malondialdehyde (MDA) and activities of N-acetyl-beta-d-glucosaminidase (NAG), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were investigated, and the levels of reactive oxygen species (ROS) and apoptosis and the pathological changes were also observed. In addition, the effects of 1 mmol/kg tea polyphenols (TP) and 0.04 mmol/kg schisandrin B (Sch B) were studied at 8.8 μmol/kg HgCl₂. It was observed that the levels of Hg, BUN, urine protein, GSH, and MDA and activities of NAG, ALP, and LDH increased significantly; the activities of SOD and GSH-Px decreased significantly; the levels of ROS and apoptosis increased obviously; and many pathological changes occurred dose-dependently in the HgCl₂ injection groups. Further investigation indicated that pretreatment with TP and Sch B significantly reversed the toxic effects of HgCl₂. These results suggested that TP and Sch B might antagonize the nephrotoxicity caused by HgCl₂ exposure.     

Effects of mercury and cadmium on the activities of antioxidative enzymes in the seedlings ofPhaseolus aureus

Phaseolus aureus Roxb. was exposed to HgCl₂ and Cd(NO₃)₂ either at the germination stage in concentration 0.5, 5 and 25 μM for 48 and 96 h, or at the seedling stage (5^th day of germination) in concentration 0.5, 5 and 20 μM for 6, 24 and 48 h. The germination and the growth of roots (germination stage treatment) were less in Hg than in Cd treatment. The seedlings (seedling stage treatment) were, however, more susceptible to Cd than Hg. Both root and leaf tissues of the plant treated at the germination stage showed enhanced lipid peroxidation and activities of the antioxidative enzymes (catalase, guaiacol peroxidase and ascorbate peroxidase), except the catalase in leaf in 25 μM Cd treatment. At seedling stage the content of malondialdehyde increased significantly only in the leaf tissue, during 6 h exposure. The activities of all the enzymes exhibited an increasing trend in both the tissue of the seedlings, particularly the leaf, at least after 24 and 48 h, except the catalase whose activity declined in response to Cd. Active involvement of the guaiacol and ascorbate peroxidases, rather than catalase, in scavenging cellular H₂O₂ was indicated. It was concluded that the two metals had little primary damaging effect on membranes.     

Effects of Lycopene and Proanthocyanidins on Hepatotoxicity Induced by Mercuric Chloride in Rats

To evaluate the protective potential of lycopene (Lyc) and proanthocyanidins (PCs) against mercuric chloride (HgCl₂)-induced hepatotoxicity, the study focused on the mechanism of oxidative stress. Firstly, the rats were subcutaneously (s.c.) injected with 0, 2.2, 4.4, and 8.8 μmol/kg HgCl₂. Additionally, 40 mg/kg Lyc and 450 mg/kg PCs were given to the rats intragastrically (i.g.) before exposure to 8.8 μmol/kg HgCl₂. Then, body weight, liver weight coefficient, mercury (Hg) contents, histological feature, ultrastructure, apoptosis, reactive oxygen species (ROS), glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA) in the liver were measured. Lactate dehydrogenase (LDH) and alanine transaminase (ALT) in serum were determined. After exposure to different concentrations of HgCl₂,it was found that Hg contents, pathological and ultrastructure injury, activities of LDH and ALT, apoptosis, and levels of ROS, GSH, and MDA increased and the activities of SOD and GSH-Px decreased in a concentration-dependent manner. Further investigation found that pretreatment with Lyc and PCs inhibited ROS production, protected antioxidant enzymes, and reversed hepatotoxicity. We concluded that Lyc and PCs had hepatoprotective effects on HgCl₂-induced toxicity by antagonizing oxidative stress in rat liver.     

Effect of acute administration of mercuric chloride on the disposition of copper, zinc, and iron in the rat

The present study was designed to investigate the effect of mercuric chloride administration on copper, zinc, and iron concentrations in the liver, kidney, lung, heart, spleen, and muscle of rats. The results showed that after dose and time exposure to mercuric chloride, the concentration of mercury in the six tissues was significantly elevated. Data showed that there were no interaction between mercury and tissue iron. There was a considerable elevation of the content of copper in the kidney and liver. The most significant changes in the copper concentration took place in the kidneys. About a twofold increase in the copper content of the kidney was noted after exposure to mercuric chloride (3 mg and 5 mg/kg). Only slight elevations in the copper content occurred in the liver, especially in high dose and longer exposure time. In the remaining organs, the copper content was not changed significantly (p>0.05). The most significant changes in the zinc concentration took place in liver, kidney, lung, and heart (5 mg/kg). Marked changes in kidney zinc concentrations were observed at any of the specified doses. Zinc concentrations were significantly increased in kidney of rats sacrificed 9–48 h after sc injection of HgCl₂ (5 mg/kg); in liver obtained from rats at 18, 24, or 48 h after injection; and in lung after 24 or 48 h of treatment. The heart and spleen zinc concentrations were elevated at 24 and 48 h after injection of HgCl₂ (5 mg/kg), respectively. The results of this study implicate that effects on copper and zinc concentrations of the target tissues of mercury may play an important role in the pathogenesis of acute mercuric chloride intoxication.     

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg²⁺) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl₂), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg²⁺ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl₂ and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg²⁺-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C and 0.5 μM of HgCl₂ solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.     

A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film

A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 μm thick whereas the bottom layer of copolymer plus E. coli cells was 30 μm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl₂ in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl₂. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at −20°C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl₂. Received 05 November 1998/ Accepted in revised form 08 April 1999     

Mercury bioremediation by mercury accumulating Enterobacter sp. cells and its alginate immobilized application

The effective microbial remediation of the mercury necessitates the mercury to be trapped within the cells without being recycled back to the environment. The study describes a mercury bioaccumulating strain of Enterobacter sp., which remediated mercury from the medium simultaneous to its growth. The transmission electron micrographs and electron dispersive X-ray analysis revealed the accumulation of remediated mercury as nano-size particles in the cytoplasm as well as on the cell wall. The Enterobacter sp. in the present work was able to accumulate mercury, without being engineered in its native form. The possibility of recovering the accumulated mercury from the cells is also indicated. The applicability of the alginate immobilized cells in removing mercury from synthetic and complex industrial effluent in a batch mode was amply demonstrated. The initial load of 7.3 mg l⁻¹ mercury in the industrial effluent was completely removed in 72 h. The cells immobilized in calcium alginate were similarly effective in the complete removal of 5 mg l⁻¹ HgCl₂ of mercury from the synthetic effluent in less than 72 h. The immobilized cells could be reused for multiple cycles.     

Zinc and N-acetylcysteine modify mercury distribution and promote increase in hepatic metallothionein levels

This study investigated the ability of zinc (Zn) and N-acetylcysteine (NAC) in preventing the biochemical alterations caused by mercury (Hg) and the retention of this metal in different organs. Adult female rats received ZnCl₂ (27 mg/kg) and/or NAC (5 mg/kg) or saline (0.9%) subcutaneously and after 24 h they received HgCl₂ (5 mg/kg) or saline (0.9%). Twenty-four hours after, they were sacrificed and analyses were performed. Hg inhibited hepatic, renal, and blood δ-aminolevulinic acid dehydratase (δ-ALA-D) activity, decreased renal total thiol levels, as well as increased serum creatinine and urea levels and aspartate aminotransferase activity. HgCl₂-exposed groups presented an important retention of Hg in all the tissues analyzed. All pre-treatments demonstrated tendency in preventing hepatic δ-ALA-D inhibition, whereas only ZnCl₂ showed this effect on blood enzyme. Moreover, the combination of these compounds completely prevented liver and blood Hg retention. The exposure to Zn and Hg increased hepatic metallothionein levels. These results show that Zn and NAC presented promising effects against the toxicity caused by HgCl₂.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl<Subscript>2</Subscript>

Mercuric chloride (HgCl₂) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl₂ on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 μM HgCl₂, either in the absence or in the presence of 60 μM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl₂ exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl₂ and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl₂.     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Impairment of Spermatogenesis in Rats by Mercuric Chloride: Involvement of Low 17β-Estradiol Level in Induction of Acute Oxidative Stress

Mercuric chloride (HgCl₂) has been shown to affect the male reproductive organs, and oxidative stress has been linked with hypospermatogenesis and with male infertility. However, the specific mode of impairment of spermatogenesis during HgCl₂ exposure has not yet been clarified fully. Because of the involvement of 17β-estradiol (E2) in the male reproductive tract and its putative role on spermatogenesis, the present study aimed to investigate the possibility that HgCl₂-induced oxidative stress-mediated modulation of the E2 level exerts adverse effects on testicular steroidogenic and gametogenic activities. HgCl₂ treatment at 50 and 100 ppm for 90 days by continuous oral administration in the drink water resulted in significant dose-dependent fashion decrease in serum and testicular E₂ levels and an increase in testicular testosterone levels in dose-dependent manner, without statistical alteration in serum testosterone level among HgCl₂ exposed groups compared to the control. Cauda epididymal sperm count and motility were decreased significantly (p < 0.01), in a dose-dependent manner, in the HgCl₂-treated groups, and qualitative examination revealed inhibition of spermatogenesis and the preferential loss of maturing and elongated spermatids. The seminiferous tubules were dilated in treated animals. When compared to the control, increase in lipid peroxidation due to toxic effects of HgCl2 was accompanied by significant reduction (p < 0.01) in antioxidant enzymes activities, superoxide dismutase, catalase, and glutathione peroxidase of testes, implicating the presence of oxidative tissue damage. Furthermore, these tissue injuries caused functional impairment as evidenced with testicular elevated activity of lactate dehydrogenase. Unless oxidative stress can lead to cancer development, testis’ tumor markers as beta human chorionic gonadotropin and alpha-fetoprotein levels have shown no significant differences in the HgCl₂-exposed group compared with respect to the control. Large quantities of metal accumulated in the testis tissue are in agreement with the testis-activity failure verified in this tissue. These findings suggest that a decrease in E2 level after mercury exposure may render testis more susceptible to oxidative damage leading to its functional inactivation, thus providing new dimension to mechanisms underlying heavy metal-induced male infertility.