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Archives of Toxicology (v.80, #10)

Evaluation of the use of salivary lead levels as a surrogate of blood lead or plasma lead levels in lead exposed subjects by Fernando Barbosa Jr; Maria Heloísa Corrêa Rodrigues; Maria R. Buzalaf; Francisco J. Krug; Raquel F. Gerlach; José Eduardo Tanus-Santos (pp. 633-637).

We conducted a study to evaluate the use of parotid salivary lead (Pb-saliva) levels as a surrogate of the blood lead (Pb-B) or plasma lead levels (Pb-P) to diagnose lead exposure. The relationship between these biomarkers was assessed in a lead exposed population. Pb-saliva and Pb-P were determined by inductively coupled plasma mass spectrometry, while in whole blood lead was determined by graphite furnace atomic absorption spectrometry. We studied 88 adults (31 men and 57 women) from 18 to 60 years old. Pb-saliva levels varied from 0.05 to 4.4 μg/l, with a mean of 0.85 μg/l. Blood lead levels varied from 32.0 to 428.0 μg/l in men (mean 112.3 μg/l) and from 25.0 to 263.0 μg/l (mean 63.5 μg/l) in women. Corresponding Pb-Ps were 0.02–2.50 μg/l (mean 0.77 μg/l) and 0.03–1.6 μg/l (mean 0.42 μg/l) in men and women, respectively. A weak correlation was found between Log Pb-saliva and Log Pb-B (r=0.277, P<0.008), and between Log Pb-saliva and Log Pb-P (r=0.280, P=0.006). The Pb-saliva/Pb-P ratio ranged from 0.20 to 18.0. Age or gender does not affect Pb-saliva levels or Pb-saliva/Pb-P ratio. Taken together, these results suggest that salivary lead may not be used as a biomarker to diagnose lead exposure nor as a surrogate of plasma lead levels at least for low to moderately lead exposed population.

Cadmium restores in vitro splicing activity inhibited by zinc-depletion by Myeong Jin Lee; Hitoshi Ayaki; Junko Goji; Keiko Kitamura; Hisahide Nishio (pp. 638-643).

Zinc (Zn)-depletion inhibits the second step of RNA splicing, namely exon-ligation. To investigate the effects of cadmium (Cd) and other metal ions on RNA splicing inhibited by Zn-depletion, we measured in vitro splicing activities in the presence of these metals. Zn-depletion in the splicing reaction mixture was achieved by addition of a Zn-chelator, 1,10-phenanthroline. Cd(II) at 1, 5 and 10 μM restored the splicing activity to 2, 24 and 72% of that in the control reaction mixture, while higher concentrations of Cd(II) decreased the splicing activity, and more than 50 μM Cd(II) showed a complete absence of spliced products. Hg(II) also restored the splicing activity, albeit to a lesser extent, since 5 and 10 μM Hg(II) restored the splicing activity to 3 and 4% of the control value. The other metal ions examined in this study, Co(II), Cu(II), Mg(II) and Mn(II), did not show any restoration of the splicing activity. We concluded that Cd(II) could restore the in vitro splicing activity inhibited by Zn-depletion, although higher concentrations of Cd(II) prevented progress of the RNA splicing reaction. These results suggest that Cd(II) has a bifunctional property regarding RNA splicing, and is stimulatory at low concentrations and inhibitory at high concentrations.

Keywords: Cadmium; In vitro splicing; Restoration; Zinc-depletion

Penetration of β-naphthylamine and o-toluidine through human skin in vitro by Lars Lüersen; Tanja Wellner; Holger M. Koch; Jürgen Angerer; Hans Drexler; Gintautas Korinth (pp. 644-646).

Several aromatic amines (AAs) are known to be carcinogens for humans. AAs are considered to be substantially absorbed through the skin. However, the database for dermal absorption of AAs in general is limited and no specific studies on dermal absorption of β-naphthylamine (BNA) and o-toluidine (OT) have been published. In the present study using diffusion cells, we investigated dermal penetration of BNA and OT through human skin. We have demonstrated that both AAs penetrate through human skin fast (lag time: ∼1.2 vs. 0.8 h) and in high percentages (54 vs. 50%, respectively, of the applied dose within 24 h). A skin notation is therefore justified for these substances.

Keywords: Dermal absorption; Diffusion cell; Aromatic amines; β-Naphthylamine; o-Toluidine

Toxicokinetics of bisphenol A in pregnant DA/Han rats after single i.v. application by S. Moors; P. Diel; G. H. Degen (pp. 647-655).

Bisphenol A (BPA) is an important chemical in the production of epoxy resins and polycarbonate plastics, and basic monomers which are used for a variety of applications. Consumer exposure to BPA may be possible from migration of BPA from dental sealants or from polycarbonate or epoxy-lined food and drink containers. BPA is known to act as weak estrogen mimic in rodents, and there is a concern of adverse endocrine effects, especially from prenatal exposure to this potential ‘endocrine disruptor’. To address this concern, we have studied the disposition and transplacental transfer of BPA in pregnant DA/Han rats on day 18 of gestation. The BPA concentrations were determined by GC/MS analysis in maternal blood, maternal organs (liver, kidney, uterus), placenta and fetuses (fetal liver and residual tissues) at different time-points (5–360 min) after intravenous administration of 10 mg BPA/kg body weight. Total BPA (aglycone and conjugates) was analyzed in all tissue samples after enzymatic hydrolysis and liquid/liquid extraction; in maternal plasma, total BPA and BPA aglycone were analyzed in parallel samples (with/without hydrolysis). Soon (5 min) after the i.v. injection a mean total BPA concentration of 3.8 μg/ml was found in maternal plasma; it declined in the first 2 h to 0.7 μg/ml. Early after injection, the majority of circulating BPA (almost 80%) was still in the aglycone form, but, metabolism by phase II enzymes decreased the BPA aglycone concentration to 0.3 μg/ml after 2 h. Despite this efficient conjugation, BPA was rapidly distributed in the organism: In well perfused organs peak concentrations for total BPA were attained 20–30 min after intravenous administration, with mean values of about 9.7 μg/g in maternal liver, 8.6 μg/g in kidneys, and 6.2 μg/g in the uterus. The peak values in other tissues were lower, with 4.0 μg/g for placenta, 3.3 μg/g for fetal liver, and 2.4 μg/g for residual fetus homogenate. The BPA levels in all tissues thereafter declined more or less in parallel with those in maternal blood. The rather similar concentration time course in placenta and fetal liver indicates that BPA is readily transferred across the placenta of DA/Han rats to the fetus. Our data on BPA disposition in DA/Han rats are discussed in the context of other kinetic studies with BPA in pregnant rats, and in relation to the previous results from our laboratory (Degen et al. Arch Toxicol 76:23–29, 2002a, b, c) demonstrating comparable transplacental transfer of daidzein, a phytoestrogen that accounts for a significant portion of total human exposure to potential endocrine disruptors.

Keywords: Bisphenol A; Environmental estrogen; Plasma and tissue level; Transplacental transfer

A dose-response study on the estrogenic activity of benzophenone-2 on various endpoints in the serum, pituitary and uterus of female rats by Christiane Schlecht; Holger Klammer; Wolfgang Wuttke; Hubertus Jarry (pp. 656-661).

The tetrahydroxylated biphenyl-ketone 2,2′,4,4′-tetrahydroxybenzophenone (BP2), one of twelve benzophenone-derived UV-filters, is used in cosmetic products and in packaging materials to protect these products from light induced damage. Recently published studies showed that BP2 exerts estrogenic activity; thus, it is an endocrine active chemical. We present data from a pharmacodynamic dose-response experiment with five dosages of BP2 applied per gavage to adult ovariectomized (ovx) rats for 5 days. Estradiol-valerate (E2) served as a control compound. The uterotrophic assay, proposed by the OECD, was modified to have a broader view on endocrine activity outside the urogenital tract to prevent that undesirable actions in other organs regulated by estrogens are missed. The gene expression levels of marker genes of estrogenic action were measured by semi-quantitative RT-PCR. Metabolic parameters were assessed by determination of the serum concentrations of leptin, cholesterol, high- and low-density lipoproteins, and triglycerides in the serum. Administration of BP2 at dosages of 10–1,000 mg/kg bodyweight led to changes of these parameters comparable to the changes in the E2 group with 0.6 mg/kg bodyweight. For the observed estrogenic activities of BP2, the “no observed adverse effect levels” were determined. Additionally, the data were further analyzed using the benchmark approach. If BP2 is transcutaneously absorbed in the human, the obtained threshold values would suggest refraining from the further use of BP2 as UV-filter in cosmetic products although additional toxicological studies should be conducted to clarify possible adverse effects.

Keywords: 2,2′,4,4′-tetrahydroxybenzophenone; BP2; Multi-organic risk assessment; Estradiol; Estrogen receptor; Benchmark approach; Rat

Molecular targets against mustard toxicity: implication of cell surface receptors, peroxynitrite production, and PARP activation by Ahmet Korkmaz; Hakan Yaren; Turgut Topal; Sukru Oter (pp. 662-670).

Despite many years of research into chemical warfare agents, cytotoxic mechanisms induced by mustards are not well understood. Reactive oxygen and nitrogen species (ROS and RNS) are likely to be involved in chemical warfare agents induced toxicity. These species lead to lipid peroxidation, protein oxidation, and DNA injury, and trigger many pathophysiological processes that harm the organism. In this article, several steps of pathophysiological mechanisms and possible ways of protection against chemical warfare agents have been discussed. In summary, pathogenesis of mustard toxicity is explained by three steps: (1) mustard binds target cell surface receptor, (2) activates intracellular ROS and RNS leading to peroxynitrite (ONOO⁻) production, and (3) the increased ONOO⁻ level damages organic molecules (lipids, proteins, and DNA) leading to poly(adenosine diphosphate-ribose) polymerase (PARP) activation. Therefore, protection against mustard toxicity could also be performed in these ways: (1) blocking of cell surface receptor, (2) inhibiting the ONOO⁻ production or scavenging the ONOO⁻ produced, and (3) inhibiting the PARP, activated by ONOO⁻ and hydroxyl radical (OH^•) induced DNA damage. As conclusion, to be really effective, treatment against mustards must take all molecular mechanisms of cytotoxicity into account. Combination of several individual potent agents, each blocking one of the toxic mechanisms induced by mustards, would be interesting. Therefore, variations of combination of cell membrane receptor blockers, antioxidants, nitric oxide synthase inhibitors, ONOO⁻ scavengers, and PARP inhibitors should be investigated.

Keywords: Nitrogen mustard; Sulfur mustard; Nitric oxide; Peroxynitrite; PARP

Expressions of N-methyl-d-aspartate receptors NR2A and NR2B subunit proteins in normal and sulfite-oxidase deficient rat’s hippocampus: effect of exogenous sulfite ingestion by Oktay Hasan Öztürk; Vural Küçükatay; Zafer Yönden; Aysel Ağar; Hüseyin Bağci; Namik Delibaş (pp. 671-679).

Sulfites whether ingested or produced through the sulfur-containing amino acids metabolism of the animal are very active molecules and can cause cellular toxicity. Sulfite oxidase (SOX), a heme- and molybdenum containing mitochondrial enzyme, prevents mammalian cells from adverse effects of sulfite toxicity by metabolizing sulfite to sulfate. The present study was aimed to investigate effect of sulfite on the N-methyl-d-aspartate (NMDA) receptor (NMDAR) NR2A and NR2B subunits in hippocampus of normal and SOX-deficient rats. Rats were divided into four groups; (1) control group, which was given rat chow and tap water ad libitum (C), (2) sulfite group, treated with sulfite (25 mg/kg) in drinking water and commercial rat chow ad libitum (S), (3) SOX-deficient group, maintained on high-W/Mo-deficient regimen to produce SOX deficiency (D), and (4) SOX-deficient + sulfite group (DS), prepared as those in the third group and were afterwards given sulfite (25 mg/kg) additionally. Whole treatment schedule were continued for 6 weeks. Sulfite treatment caused a decrease of NR2A and NR2B subunits of the NMDAR in hippocampus of rats in S and DS groups. Interestingly, similar decrement was observed in D group, probably due to increased endogen sulfite production. In summary, the results indicated that feeding sulfite to the rats may cause down-regulation of NMDARs by degrading NR2A and NR2B subunits of it, which may be considered as a neuro-compensatory mechanism.

Cadmium and cisplatin damage erythropoietin-producing proximal renal tubular cells by Hyogo Horiguchi; Etsuko Oguma; Fujio Kayama (pp. 680-686).

The concomitant manifestations of proximal renal tubular dysfunction and anemia with erythropoietin (Epo) deficiency observed in chronic cadmium (Cd) intoxication, such as Itai-itai disease, suggest a close local correlation between the Cd-targeted tubular cells and Epo-producing cells in the kidney. Therefore, we investigated the local relationship between hypoxia-induced Epo production and renal tubular injury in rats injected with Cd at 2 mg/kg twice a week for 8 months. Anemia due to insufficient production of Epo was observed in Cd-intoxicated rats. In situ hybridization detected Epo mRNA expression in the proximal renal tubular cells of hypoxic rats without Cd intoxication, and the Cd-intoxicated rats showed atrophy of Epo-expressing renal tubules and replacement of them with fibrotic tissue. A single dose of cisplatin at 8 mg/kg, which can induce clinical manifestations similar to those of Cd including renal tubular damage along with Epo-deficient anemia, resulted in Epo-expressing renal tubule destruction on day 4. These data indicate that Cd and cisplatin would induce anemia through the direct injury of the proximal renal tubular cells that are responsible for Epo production.

Keywords: Cadmium; Erythropoietin; Anemia; Kidney; Cisplatin

Paraquat-induced gene expression in rat kidney by Masafumi Tomita; Toshiko Okuyama; Hironobu Katsuyama; Takaki Ishikawa (pp. 687-693).

Paraquat, one of the most widely used herbicides, is highly toxic to humans and animals. There is much information regarding its toxic effects on the lungs, but less is known about its toxicity in other organs. Paraquat is thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. We investigated the effects of paraquat on gene expression in the kidneys of rats treated with paraquat using a DNA array system, and the gene up-regulation observed was confirmed by quantitative real-time RT-PCR. Rats were sacrificed at 3, 24 h after the first injection (20 mg/kg), and at 3 h after the second injection. Expression of six genes had increased significantly by 3 h after the first injection: metallothionein-1 (MT-1), phosphoenolpyruvate carboxykinase, Na/K-transporting ATPase β1 subunit, glutamate oxaloacetic transaminase, glutathione-S-transferase, and heme oxygenase-1 (HO-1). The transcription levels of MT-1 and HO-1 showed the biggest increases, but the increases did not continue until 24 h after injection, and the second injection had less effect than the first. Up-regulation of MT-1 and HO-1 mRNA levels was confirmed at the protein level. We observed a paraquat-induced increase of these proteins at 3 h post-injection, whereas this level did not continue until 24 h, as observed in RNA levels. The MT-1 protein in kidneys had been consumed. In addition, the protein level due to the second injection did not increase to the same level as that due to the first injection. These results suggest that protection against paraquat injury is mediated by induction of expression of some genes, and suppression on the induction of MT-1 and HO-1 may explain the injury observed due to paraquat intake. This is the first report of inducible pathways of defense against paraquat-induced oxidative stress in the kidney.

Keywords: Paraquat; Toxicity; Oxidative stress; Gene expression; Kidney

Possible involvement of oxidative stress in dicyclanil-induced hepatocarcinogenesis in mice by Mitsuyoshi Moto; Takashi Umemura; Miwa Okamura; Masako Muguruma; Tadashi Ito; Mailan Jin; Yoko Kashida; Kunitoshi Mitsumori (pp. 694-702).

Our previous study suggested the possibilities that dicyclanil (DC), a nongenotoxic carcinogen, produces oxidative stress in the liver of the two-stage hepatocarcinogenesis model of mice and the stress induced probably causes secondary oxidative DNA damage. However, clear evidences demonstrating the relationship between DC-induced hepatocarcinogenesis, oxidative stress, and oxidative DNA damage have not been obtained. To clarify the relationship, further investigations were performed in the liver of the partially hepatectomized (PH) mice maintained on diet containing 1,500 ppm of DC for 13 and 26 weeks after intraperitoneal injection of dimethylnitrosamine (DMN). Significant increases in mRNA expressions of some metabolism- and oxidative stress-related genes with a formation of γ-glutamyltranspeptidase (GGT) positive foci were observed in the DMN + DC + PH group by the treatment of DC for 13 and 26 weeks. The levels of 8-hydroxy-deoxyguanosine (8-OHdG) in the liver DNA also significantly increased in mice of the DMN + DC + PH group at weeks 13 and 26 and mice given DC alone for 26 weeks. The in vitro measurement of reactive oxygen species (ROS) generation from the mouse liver microsomes showed a significant increase of ROS production in the presence of DC. These results suggest that DC induces oxidative stress which is probably derived from its metabolic pathway, partly, and support our previous speculation that oxidative stress plays one of the important roles in the DC-induced hepatocarcinogenesis in mice.

Keywords: Dicyclanil; Hepatocarcinogenesis; Oxidative stress; Nongenotoxic carcinogen; Mouse

Effects of subchronic exposure to styrene on the extracellular and tissue levels of dopamine, serotonin and their metabolites in rat brain by F. Gagnaire; M. Chalansonnet; N. Carabin; J.-C. Micillino (pp. 703-712).

At present, there is controversy over the neurotoxic potential of styrene. Several epidemiological and clinical studies have shown that styrene exposure causes alterations of central nervous system functions in humans. Neurotransmitters have been implicated in the pathogenesis of styrene neurotoxicity in rodents. Several studies carried out on postmortem brain tissue suggest that styrene may alter dopaminergic neurotransmission in rabbit or rat brain. Moreover, in vitro studies suggest that both styrene and styrene oxide inhibit the uptake of dopamine (DA) in purified synaptic vesicles prepared from rat brain striata. To date, biochemical studies on animals have explored global tissue levels of neurotransmitters with sub-acute exposures to styrene. However, extracellular levels of neurotransmitters are more closely related to behaviour than are global tissue levels. The present study determined changes in the extracellular concentrations of DA, serotonin (5-HT) and their acid metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), in striatal dialysates from freely moving adult male rats after exposure to 750 and 1,000 ppm styrene, 6 h per day, 5 days per week for 4 weeks. We also determined the concentrations of DA, 5-HT and their acid metabolites in striatum, nucleus accumbens and prefrontal cortex obtained postmortem from similarly exposed rats. Exposure to 1,000 ppm of styrene caused a significant decrease in extracellular acid metabolite concentrations. Tissue levels of acid metabolites were also decreased to a lesser extent. The effects were observed 72 h after discontinuing exposure but had vanished 17 days later. There was no change in DA or 5-HT concentrations either in the dialysates or tissues. Exposure to 750 ppm styrene caused no changes in the concentrations of DA, 5-HT and their acid metabolites either in the dialysates or tissues. The possibility that the effect of styrene is mediated by monoamine oxidase (MAO) inhibition is discussed.

Keywords: Styrene; Dopamine; Serotonin; Microdialysis; Rat

Loss of imprinting of the insulin-like growth factor 2 and the H19 gene in testicular seminomas detected by real-time PCR approach by Sebastian Stier; Thomas Neuhaus; Peter Albers; Nicolas Wernert; Elisabeth Grünewald; Randolf Forkert; Hans Vetter; Yon Ko (pp. 713-718).

IGF2 and H19 are imprinted genes in normal human tissue, but many studies have observed a loss of imprinting (LOI) of these genes in tumors as an epigenetic alteration of the DNA, that leads to a biallelic expression predisposing cells to carcinogenesis and tumor growth. The aim of this study was to test the reliability of LightCycler™-assisted Real-time PCR in detecting LOI of IGF2 and H19 in 39 patients with testicular germ cell tumors by comparing these results with the analysis generated by the golden standard restriction fragment length polymorphism (RFLP). With LightCycler™-assisted Real-time PCR for IGF2 44% and for H19 49% of the patients were found to be heterozygous. This was consistent with the results obtained by RFLP, but surprisingly RFLP failed in more than 7% of the patients. In detecting LOI (for IGF2 in 41% and for H19 in 68% of the informative patients) the approach by RFLP was superior, since the results derived from LightCycler™-assisted Real-time PCR showed reliable results in 76 and 10% of the samples concerning IGF2 and H19, respectively. Again, no discrepancy between the results obtained by the two methods occurred. In sum, LightCycler™-assisted Real-time PCR is a sufficiently working approach for the rapid and reliable detection of heterozygosity of IGF2 or H19 gene and identification of LOI of IGF2 and thus may be helpful in conducting large epidemiological studies. However, for the identification of LOI of the H19 gene in this cohort it possesses only restrictive use.

Keywords: Loss of imprinting; IGF2; H19; Testicular seminomas; Real-time PCR

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