DJ-1 and Parkin modulate dopamine-dependent behavior and inhibit MPTP-induced nigral dopamine neuron loss in mice.

Parkin-deficient animals exhibit mitochondrial degeneration and increased oxidative stress vulnerability, and both mice and flies lacking DJ-1 are hypersensitive to environmental toxins associated with Parkinson's disease (PD). We used recombinant adeno-associated virus (AAV) gene transfer to study the influence of DJ-1 and Parkin on the dopaminergic system of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, a model for sporadic PD. After MPTP lesioning, significantly more dopamine neurons survived in the virus-injected substantia nigra of the AAV-DJ-1 and AAV-Parkin mice when compared with AAV-enhanced green fluorescent protein injected controls. Protection at the neuronal level was supported by increased amphetamine-induced contralateral turning behavior. Normal mice expressing DJ-1 showed apomorphine-induced ipsilateral turning, suggesting a hyporesponsiveness of striatal dopamine D1 receptors in the DJ-1-expressing hemisphere. MPTP drastically reduced dopamine to 19% of normal levels and neither DJ-1 nor Parkin protected against MPTP-induced catecholamine loss under these conditions. Our results show that Parkin and DJ-1 inhibit dopamine neuron death and enhance amphetamine-induced dopaminergic function in a mouse model of idiopathic PD. However, DJ-1 overexpression also reduced postsynaptic dopamine receptor responses in normal mice. These results warrant further exploration of DJ-1 and Parkin gene therapy for PD, although a better understanding of their effects on behavior and dopamine neurotransmission is required before these proteins can be safely used.     

Renal organic anion transporters OAT1 and OAT3 mediate the cellular accumulation of 5-sulfooxymethylfurfural, a reactive, nephrotoxic metabolite of the Maillard product 5-hydroxymethylfurfural

5-Hydroxymethylfurfural (HMF) is formed when sugars are acidified or heated. It is present at high levels in numerous foods. HMF is inactive in standard genotoxicity tests, but can be metabolized to a chemically reactive intermediate, 5-sulfooxymethylfurfural (SMF), which is mutagenic and carcinogenic. We recently found that direct parental administration of SMF to mice leads to abundant acute necrosis and proteinaceous casts in the proximal tubules as the dominating toxicological effect. Since proximal tubule cells actively mediate the excretion of many organic anions, we hypothesized that transporter-mediated uptake of SMF into the cells could be the reason for this selective organotoxicity. To test this hypothesis, we used human embryonic kidney (HEK293) cells stably expressing human (h) OAT1 or OAT3. SMF was a competitive inhibitor of p-aminohippurate uptake by hOAT1 and estrone sulfate uptake by hOAT3 with K_i values of 225 μM and 1.5 mM, respectively. Moreover, the initial rates of SMF uptake were 5.2- and 3.1-fold higher in cells expressing hOAT1 and hOAT3, respectively, than in control HEK293 cells. Likewise, the sensitivity of hOAT1- and hOAT3-expressing cells to SMF cytotoxicity was significantly higher than that of control cells, and was reduced by addition of probenecid, an inhibitor of OATs. Taken together, these results indicate that OAT1 and OAT3 mediate the uptake of SMF into proximal tubule cells and thereby may be involved in SMF-induced nephrotoxicity.     

Impaired mitochondrial dynamics and function in the pathogenesis of Parkinson's disease

Parkinson's disease (PD), the most frequent movement disorder, is caused by the progressive loss of the dopamine neurons within the substantia nigra pars compacta (SNc) and the associated deficiency of the neurotransmitter dopamine in the striatum. Most cases of PD occur sporadically with unknown cause, but mutations in several genes have been linked to genetic forms of PD (α-synuclein, Parkin, DJ-1, PINK1, and LRRK2). These genes have provided exciting new avenues to study PD pathogenesis and the mechanisms underlying the selective dopaminergic neuron death in PD. Epidemiological studies in humans, as well as molecular studies in toxin-induced and genetic animal models of PD show that mitochondrial dysfunction is a defect occurring early in the pathogenesis of both sporadic and familial PD. Mitochondrial dynamics (fission, fusion, migration) is important for neurotransmission, synaptic maintenance and neuronal survival. Recent studies have shown that PINK1 and Parkin play crucial roles in the regulation of mitochondrial dynamics and function. Mutations in DJ-1 and Parkin render animals more susceptible to oxidative stress and mitochondrial toxins implicated in sporadic PD, lending support to the hypothesis that some PD cases may be caused by gene–environmental factor interactions. A small proportion of α-synuclein is imported into mitochondria, where it accumulates in the brains of PD patients and may impair respiratory complex I activity. Accumulation of clonal, somatic mitochondrial DNA deletions has been observed in the substantia nigra during aging and in PD, suggesting that mitochondrial DNA mutations in some instances may pre-dispose to dopamine neuron death by impairing respiration. Besides compromising cellular energy production, mitochondrial dysfunction is associated with the generation of oxidative stress, and dysfunctional mitochondria more readily mediate the induction of apoptosis, especially in the face of cellular stress. Collectively, the studies examined and summarized here reveal an important causal role for mitochondrial dysfunction in PD pathogenesis, and suggest that drugs and genetic approaches with the ability to modulate mitochondrial dynamics, function and biogenesis may have important clinical applications in the future treatment of PD.     

Follow-up in non-small-cell lung cancer

The major impetus for structured follow-up after primary treatment of localized non-small-cell lung cancer is both early detection and re-treatment of recurrent disease with a curative intent, and early detection and treatment of a secondary primary tumor. In patients with lung cancer, the significance of intensive follow-up is still under debate.     

Probenecid,an inhibitor of transmembrane organic anion transporters,alters tissue distribution of DNA adducts in 1-hydroxymethylpyrene-treated rats

1-Methylpyrene (1-MP), an abundant alkylated polycyclic aromatic hydrocarbon, is activated by side-chain hydroxylation to 1-hydroxymethylpyrene (1-HMP) and subsequent sulfo-conjugation to electrophilic 1-sulfooxymethylpyrene (1-SMP). In rats, this activation mainly occurs in liver. 1-SMP may react with hepatic DNA or be exported into the blood circulation to reach other tissues, in particular kidneys. Findings with recombinant cell lines suggest that renal 1-SMP uptake proceeds via organic anion transporters (OATs). Here, we tested the hypothesis that probenecid, a characteristic OAT inhibitor, interferes with kidney damage brought about by 1-SMP formed in rats. 1-HMP was administered intraperitoneally to 30 rats, half of which were co-treated with probenecid. The tissue distribution of DNA adducts was analyzed using ³²P-postlabeling and isotope dilution LC–MS/MS for the detection of the adducts N²-(1-methylpyrenyl)-2′-deoxyguanosine and N⁶-(1-methylpyrenyl)-2′-deoxyadenosine. In rats treated solely with 1-HMP, adduct levels in kidney tissue were about 3-fold and 8-fold higher than those in liver and lung, respectively. After co-treatment with probenecid, hepatic and pulmonary adduct levels were 12-fold and 4-fold elevated, respectively, whereas renal adduct levels were slightly lower compared to those of rats receiving 1-HMP alone. Moreover, serum levels of 1-SMP were increased 23-fold in animals pre-treated with probenecid. The differential effects on hepatic and pulmonary adduct levels suggest that not only renal OATs, but also additional anion transporters, e.g. those mediating the hepatic export of 1-SMP into the bile, were inhibited. Thus, transmembrane transport proteins play a crucial role in the distribution of reactive phase II metabolites, and thereby in tissue allocation of DNA adducts.     

Radio diagnosis using 125I-fibrinogen and 203Hg-thymidine

A comparative study of ¹²⁵I-fibrinogen and ²⁰³Hg-thymidine using 144 Ehrlich carcinoma-bearing mice, indicates that tumor affinity of carrier substances with covalent labeling could be better used for scintigraphic tumor diagnosis.Halogenated or possibly metallo-organic compounds offer an alternative to the metal complexes in general use up to now.     

Ethanol- or acetone-pretreatment of mice strongly enhances the bacterial mutagenicity of dimethylnitrosamine in assays mediated by liver subcellular fraction, but not in host-mediated assays

The activation of dimethylnitrosamine (DMN) to a bacterial mutagenin liver subcellular fraction and in intrasanguinous host-mediatedassays was studied, in particular the effect of pretreatmentof the animals with ethanol or acetone. Salmonella typhimuriumTA 92 was much more sensitive to DMN mutagenicity than TA 100and TA 1535 or Escherichia coli WP2 uvrA and was used for themain part of the study. Noteworthy, in part already known, featuresof the in vitro activation are the relatively low pH optimum(pH 6–6.4), the non-linear dose-mutagenic response-relationshipand the relatively high doses of DMN required for activationwith control preparations. Pretreatment of mice with ethanolor acetone greatly reduced the minimal mutagenically effectiveconcentration of DMN in the in vitro assay. Pretreatment withAroclor 1254, an inducer frequently used in mutagenicity research,showed little effect when used alone, but reduced the potentiationby acetone. The results of the host-mediated assays substantiallydiffered from those of the in vitro activation assays (a) inthe relatively low dose of DMN required for mutagenicity tooccur and (b) in the lack of potentiation by acetone- or ethanol-pretreatment.Acetone even led to a marginal decrease in mutagenicity. Asa possible explanation for this apparent discrepancy we assumethat with the in vitro system the activity of the dilute metabolizingsystem is limiting for the activation of DMN and induction thereforewill increase the mutagenicity, whereas in vivo DMN is quantitativelymetabolized in both induced and non-induced animals. The resultsshow that caution has to be taken in the interpretation fromin vitro results to the in vivo situation. In particular ourin vivo experiments do not support the hypothesis that the inductionby ethanol of an activating system with a low K_m (which wouldstrongly activate traces of DMN ingested with many foods) isone of the reasons for the increased risk of liver tumors inalcoholics.     

Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens

We have constructed Chinese hamster V79-derived cell lines (V79-rSULT1B1-A and -B) that express rat sulfotransferase 1B1 (rSULT1B1). Sulfotransferase activity towards 1-naphthol was 1020 +/- 220 pmol/min/mg cytosolic protein in V79-rSULT1B1-A cells and 57 +/- 9 pmol/ min/mg in V79-rSULT1B1-B cells. These activities were similar over 100 population doublings and at varying cell densities. Immunostaining indicated a cytoplasmatic localization of rSULT1B1. Expression usually was homogeneous within colonies but showed some variation between colonies. The level of rSULT1B1 protein in V79-rSULT1B1-B cells was similar to that in rat liver but higher than in colon mucosa. The cytotoxicity of the benzylic alcohols 4H-cyclopenta[def]chrysen-4-ol and 6-hydroxymethylbenzo-[a]pyrene was enhanced >100-fold in V79-rSULT1B1-A cells compared with SULT-deficient cells (V79p). Likewise, these compounds showed mutagenic effects (at the hprt locus) in V79-rSULT1B1-A cells starting at a concentration of 0.02 and 0.01 micro M, respectively, but were inactive in V79p cells even at a concentration of 1 micro M. The cell line with the lower expression level, V79-rSULT1B1-B, showed only marginal toxification of the compounds investigated, indicating an important role of the expression level in the test system. A thoroughly characterized mammalian cell system, including positive controls, is now available for studying rSULT1B1-mediated bioactivation of promutagens and protoxicants.     

Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase

3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity. The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2. This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella. Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.     

Directing role of organic anion transporters in the excretion of mercapturic acids of alkylated polycyclic aromatic hydrocarbons.

Excretion of mercapturic acids of a xenobiotic is a good indicator for the formation of electrophilic intermediates. However, the route of excretion, urine or feces, is important for usage of a given mercapturic acid as a biomarker in humans. In the present study we investigated the excretion routes of 1-methylpyrenyl mercapturic acid (MPMA) and 1,8-dimethylpyrenyl mercapturic acid (DMPMA) formed from the corresponding benzylic alcohols in rats. Whereas MPMA was primarily excreted in urine (72% of the total urinary and fecal level), DMPMA clearly preferred the fecal route (88%). We then examined interactions of these mercapturic acids with renal basolateral organic anion transporters (OATs) using HEK293 cells stably expressing human OAT1 and OAT3. The uptake rates of MPMA by OAT1- and OAT3-expressing cells were 2.8- and 1.7-fold, respectively, higher than that by control cells. MPMA was a competitive inhibitor of p-aminohippurate uptake by OAT1 and estrone sulfate uptake by OAT3 with K(i) values of 14.5 microM and 1.5 microM, respectively. In contrast, DMPMA was not transported by OAT1 and only modestly transported by OAT3 (1.25-fold over control). Thus, we suspect that the substrate specificities, alone or together with other factors, played a directing role in the excretion of MPMA and DMPMA. Although the mechanistic link requires verification, our results clearly show that a minute structural difference (the presence or absence of an additional methyl group in an alkylated four-ring polycyclic hydrocarbon) can strongly affect the interaction with transporter proteins and direct the excretion route of mercapturic acids.