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Archives of Toxicology (v.79, #1)

β-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure by Huoy-Rou Chang; Der-An Tsao; Hsin-Su Yu; Chi-Kung Ho (pp. 1-6).

To understanding the reversible or irreversible harm to the β-adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain β-adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and β-adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of β-adrenergic receptor using the ligand [¹²⁵I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain β-adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the β-adrenergic system of the brain is a partly reversible condition.

Keywords: Lead; β-adrenoceptor; Adenylate cyclase activity; Brain

DDT increases hepatic testosterone metabolism in rats by Adolfo Sierra-Santoyo; Manuel Hernández; Arnulfo Albores; Mariano E. Cebrián (pp. 7-12).

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-¹⁴C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2α-hydroxytestosterone (OHT), and 16α-OHT but higher 6β-OHT whereas treated females produced less 7α-OHT and AD but higher 6β-OHT and 6α-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6β-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6α-/15α-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6α-/16α-OHT and 6-dehydrotestosterone/16α-OHT ratios followed a similar tendency, with the ratio 6α-/16α-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.

Keywords: DDT; Endocrine disruption; Testosterone metabolism; Cytochrome P-450; Sex-dependent regulation

Tobacco smoke-dependent changes in cytochrome P450 1A1, 1A2, and 2E1 protein expressions in fetuses, newborns, pregnant rats, and human placenta by Piotr Czekaj; Anna Wiaderkiewicz; Ewa Florek; Ryszard Wiaderkiewicz (pp. 13-24).

Tobacco smoke (TS) was described as a mixture of numerous cytochrome P450 (P450) substrates, inducers, and inhibitors. These inducers and inhibitors may modify drug clearance and xenobiotic or endogenous metabolism affecting P450s expression. In the present study, the effect of gestation and TS on: (1) cytochrome P450 CYP1A1, CYP1A2, and CYP2E1 protein expressions, and (2) cytochrome P450-linked microsomal enzyme activities, were studied in fetal rat liver, rat, and human placenta and in newborn and adult rat hepatic and extrahepatic tissues. Non-pregnant and pregnant 4-month-old female Wistar rats were exposed to TS (500, 1,000, or 1,500 mg carbon monoxide per m³ air) in a toxicological chamber for 3 weeks (6 h daily, 5 days weekly). Human placentas were sampled from non-smoking, passive smoking, or active smoking primiparas. The efficacy of exposure was assessed by measuring urine cotinine levels. The TS-dependent inductory effect on the expression of CYP1A1 and 1A2 and related monooxygenase activities, and the inhibitory/inductory effect on CYP2E1 expression in rat tissues were observed. Pregnancy was associated with decreased levels of constitutive CYP1A1 and 2E1 in hepatic and extrahepatic tissues, TS-inducible CYP1A2 expression in the liver, and CYP1A1 expression in lungs and heart, but had no inhibitory effect on TS-inducible CYP1A1 and 2E1 expression, EROD, and P450-cooperated enzyme activities in the liver, kidney, and, in the latter case, in the heart. The presence of TS-induced CYP1A1 protein was confirmed in rat and human placenta and showed in newborn liver and lungs. CYP1A2 and 2E1 proteins were detectable in fetal rat liver. It was concluded that the expression of CYP1A1, 1A2, and 2E1, which metabolize some drugs and activate carcinogens, is controlled by age-, pregnancy-, and tissue-specific regulatory mechanisms in rats. Gestational differences in the regulation of expression of CYP1A subfamily members are not excluded. CYP1A1 and 2E1, but not CYP1A2 inductory mechanisms seem to be functional in fetal liver at day 21 of pregnancy but they appeared to be uninducible under a TS exposure. In TS-exposed pregnant females and fetuses the effects of metabolic activation of CYP1A1 and 1A2 substrates might be reduced because of lower CYP expressions or poor induction, respectively.

Keywords: Cytochrome P450; CYP1A1; CYP1A2; CYP2E1; Tobacco smoke; Pregnancy; Age

Oxidation of ethanol to acetaldehyde and free radicals by rat testicular microsomes by Leandro N. Quintans; Gerardo D. Castro; José A. Castro (pp. 25-30).

A large number of epidemiological studies evidencing that excessive alcohol consumption is associated with impaired testosterone production and testicular atrophy are available in the literature. One hypothesis to explain the deleterious action of alcohol involves the in situ biotransformation to acetaldehyde, but it strongly suggests the need to learn more about the enzymatic processes governing alcohol metabolism to acetaldehyde in different cellular fractions since limited information is available in the literature. In this article we report studies on the metabolic conversion of alcohol to acetaldehyde and to 1-hydroxyethyl radicals in rat testicular microsomal fractions. The oxidation of ethanol to acetaldehyde in rat testes microsomal fraction was mostly of enzymatic nature and strongly dependent on the presence of NADPH and oxygen. Several compounds were able to significantly decrease the production of acetaldehyde: SKF 525A; diethyldithiocarbamate; esculetin; gossypol; curcumin; quercetin; dapsone; and diphenyleneiodonium. Microsomal preparations in the presence of NADPH were also able to produce both hydroxyl and 1-hydroxyethyl free radicals. Their generation was modulated by the presence of diphenyleneiodonium, gossypol, and deferoxamine. Results show that rat microsomal fractions are able to metabolize alcohol to deleterious chemicals, such as acetaldehyde and free radicals, that may be involved in ethanol toxic effects. Enzymes involved could include CYP2E1, P450 reductase, and other enzymes having lipoxygenase- /peroxidase-like behavior.

Keywords: Alcohol; Ethanol; Acetaldehyde; 1-hydroxyethyl; Testes; Radicals; Microsomes

Transforming growth factor-β₁ is not involved in TCDD-dependent release from contact inhibition in WB-F344 cells by Peter Hoelper; Dagmar Faust; Franz Oesch; Cornelia Dietrich (pp. 31-36).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells TCDD induces a release from contact inhibition which is manifested by a two- to threefold increase in DNA synthesis when TCDD (1 nM) is applied to confluent cells. Since proliferation of epithelial cells is known to be inhibited by TGF-β, we investigated whether decreased TGF-β₁ mediates TCDD-dependent release from contact inhibition in WB-F344 cells. Expression of TGF-β (type II) receptor in WB-F344 cells was analyzed by Western blot analysis. Exposure of 0.1 ng/ml TGF-β₁ to exponentially growing WB-F344 cells resulted in a 40% decrease in DNA synthesis, which was blocked by preincubation with a neutralizing anti-TGF-β₁ antibody, indicating that the TGF-β receptor in WB-F344 cells is functionally active. Preincubation of confluent, G₁-arrested cultures with the neutralizing anti-TGF-β₁-antibody did not lead to an increase in DNA synthesis, ruling out an involvement of TGF-β₁ in mediating contact inhibition in WB-F344 cells. In accord with this, Western blot analysis revealed that protein expression of TGF-β₁ is neither upregulated in confluent cultures nor decreased after TCDD treatment. We conclude that TGF-β₁ is not involved in contact inhibition or in TCDD-dependent release from contact inhibition in WB-F344 cells.

Keywords: Contact inhibition; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; Transforming growth factor-β

Contact allergens and irritants show discrete differences in the activation of human monocyte-derived dendritic cells: consequences for in vitro detection of contact allergens by Frank Straube; Olivier Grenet; Peter Bruegger; Peter Ulrich (pp. 37-46).

In recent years test systems have been described that may be applied routinely to discriminate between contact allergens and irritants in vitro. Using human monocyte-derived dendritic cells (MoDC), this study was designed to refine the settings of a potential routine screening protocol for contact allergens and to investigate the so far poorly defined concentration dependency of contact allergen-specific effects. MoDC were generated by 6 days of culture in the presence of IL-4 and GM-CSF and were then cultured for 24 or 48 h in medium with lipopolysaccharide (LPS), contact allergens [picrylsulphonic acid (TNBS), 1-chloro-2,4-dinitrobenzene (DNCB)] or irritants [sodium dodecyl sulphate (SDS), benzalkonium chloride (BAC)] that were free of detectable endotoxin contamination. The induction of CD86 and HLA-DR expression was quantified by flow cytometry as markers for MoDC activation. LPS activation upregulated CD86 about 20-fold and HLA-DR expression about 4-fold. Compared to LPS, contact allergens had weaker effects. TNBS and DNCB induced activation marker upregulation starting slightly below the cytotoxic concentration and increasing in a dose-dependent manner. However, at partially cytotoxic concentrations, irritants also induced CD86 and HLA-DR expression, as confirmed by flow cytometry and quantitative RT-PCR. Both SDS and BAC induced activation marker expression on surviving MoDC, when more than 50% of the MoDC population had been killed by the treatment. Consequently, routine testing of unknown substances would need to quantify activation marker expression as well as cytotoxicity in parallel. In the concentration range around the lowest cytotoxic concentration, the assay may be able to discriminate between contact allergens and irritants.

Keywords: Monocyte-derived dendritic cell (MoDC); Contact allergen; Skin irritant; Dose dependency; Activation marker

The monocyte dysfunction induced by acute tetramine poisoning and corrected by continuous blood purification by Chen Yu; Zhihong Liu; Dehua Gong; Daxi Ji; Leishi Li (pp. 47-53).

The monocyte function of patients with severe tetramine poisoning and the effects of sequential hemoperfusion (HP) and continuous veno-venous hemofiltration (CVVH) on the immune status of the patients were investigated. Eleven patients with severe acute tetramine poisoning were treated with sequential HP and CVVH. The APACHE II score and Glasgow score were used to evaluate the disease status during the therapy. Blood samples were collected at 0, 2, 6, 12, 24, 48, and 72 h. Peripheral monocytes were isolated and stimulated with lipopolysaccharide (LPS) to detect the ability of monocytes to secrete tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10. The number of monocytes was counted at the same time. As expected, three patients died and the clinical manifestations were improved in the other patients. The production of cytokines (TNF-α, IL-6, and IL-10) by monocytes of patients with tetramine poisoning was much lower than normal controls (P<0.001), and was significantly increased after HP and CVVH in the survivors (TNF-α, IL-6, IL-10, P<0.05, P<0.01, P<0.05, respectively). The blood concentration of tetramine was 0.124±0.082 mg/l at prehemoperfusion and 0.080±0.055 mg/l at posthemoperfusion (P<0.05). It was concluded that there was severe damage to monocyte function in patients with tetramine poisoning, and that sequential HP and CVVH can effectively ameliorated monocyte function and eliminate tetramine from blood.

Keywords: Tetramine; Continuous veno-venous hemofiltration (CVVH); Hemoperfusion (HP); Monocyte

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells by Seishiro Hirano; Yayoi Kobayashi; Toru Hayakawa; Xing Cui; Megumi Yamamoto; Sanae Kanno; Amjad Shraim (pp. 54-61).

Clark 1 (diphenylarsine chloride) and Clark 2 (diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO³⁺) and phenylarsonic acid (PAA⁵⁺) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO³⁺ were much higher than those of PAA⁵⁺, and LC₅₀ values of PAO³⁺ and PAA⁵⁺ were calculated to be 0.295 µM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO³⁺ and PAA⁵⁺. N-Acetyl-l-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO³⁺-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA⁵⁺-exposed cells. The effect of NAC may be due to a strong affinity of PAO³⁺ to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO³⁺, but PAA³⁺ increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO³⁺ the high toxicity.

Keywords: Phenylarsine oxide; Phenylarsonic acid; Cytotoxicity; Endothelial cell; Heme oxygenase-1N-Acetyl-l-cysteine; Glutathione; Buthionine sulfoximine; Inductively coupled plasma mass spectrometry

Acknowlegements (pp. 62-62).

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Archives of Toxicology (v.79, #1)