Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Comparison of the morphological transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene in C3H10T1/2CL8 cells and characterization of the dibenzo[a,l]pyrene-DNA adducts

C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentration- response studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 microM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32P-postlabeling methods using mononucleotide 3'-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol- 13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14- epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3'-monophosphate and deoxyguanosine 3'-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti- DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE- deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.      

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

Identification of differentially expressed genes in aflatoxin B1- treated cultured primary rat hepatocytes and Fischer 344 rats

Aflatoxin B1 (AFB1), a mutagen and hepatocarcinogen in rats and humans, is a contaminant of the human food supply, particularly in parts of Africa and Asia. AFB1-induced changes in gene expression may play a part in the development of the toxic, immunosuppressive and carcinogenic properties of this fungal metabolite. An understanding of the-role of AFB1 in modulating gene regulation should provide insight regarding mechanisms of AFB1-induced carcinogenesis. We used three PCR- based subtractive techniques to identify AFB1-responsive genes in cultured primary rat hepatocyte RNA: differential display PCR (DD-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH). Each of the three techniques identified AFB1- responsive genes, although no individual cDNA was isolated by more than one technique. Nine cDNAs isolated using DD-PCR, RDA or SSH were found to represent eight genes that are differentially expressed as a result of AFB1 exposure. Genes whose mRNA levels were increased in cultured primary rat hepatocytes after AFB1 treatment were corticosteroid binding globulin (CBG), cytochrome P450 4F1 (CYP4F1), alpha-2 microglobulin, C4b-binding protein (C4BP), serum amyloid A-2 and glutathione S-transferase Yb2 (GST). Transferrin and a small CYP3A-like cDNA had reduced mRNA levels after AFB1 exposure. Full-length CYP3A mRNA levels were increased. When liver RNA from AFB1-treated male F344 rats was evaluated for transferrin, CBG, GST, CYP3A and CYP4F1 expression, a decrease in transferrin mRNA and an increase in CBG, GST, CYP3A and CYP4F1 mRNA levels was also seen. Analysis of the potential function of these genes in maintaining cellular homeostasis suggests that their differential expression could contribute to the toxicity associated with AFB1 exposure.      

Tc-99m MDP uptake in uterine leiomyoma

A 47-year-old woman with adenocarcinoma of the right breast had bone scintigraphy with Tc-99m MDP. Bone imaging did not show any metastases. However, a large area of increased tracer uptake was seen extending from the abdomen to the pelvis. Abdominal ultrasound revealed a large solid and heterogeneous mass, measuring 18 x 11 x 14.3 cm, that originated in an empty uterus. A biopsy of the surgical specimen showed a leiomyoma of the uterus.     

Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon

BACKGROUND: The mechanisms by which the inhalational general anesthetics isoflurane and xenon exert their effects are unknown. Moreover, there have been surprisingly few quantitative studies of the effects of these agents on central synapses, with virtually no information available regarding the actions of xenon. METHODS: The actions of isoflurane and xenon on gamma-aminobutyric acid-mediated (GABAergic) and glutamatergic synapses were investigated using voltage-clamp techniques on autaptic cultures of rat hippocampal neurons, a preparation that avoids the confounding effects of complex neuronal networks. RESULTS: Isoflurane exerts its greatest effects on GABAergic synapses, causing a marked increase in total charge transfer (by approximately 70% at minimum alveolar concentration) through the inhibitory postsynaptic current. This effect is entirely mediated by an increase in the slow component of the inhibitory postsynaptic current. At glutamatergic synapses, isoflurane has smaller effects, but it nonetheless significantly reduces the total charge transfer (by approximately 30% at minimum alveolar concentration) through the excitatory postsynaptic current, with the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor-mediated components being roughly equally sensitive. Xenon has no measurable effect on GABAergic inhibitory postsynaptic currents or on currents evoked by exogenous application of GABA, but it substantially inhibits total charge transfer (by approximately 60% at minimum alveolar concentration) through the excitatory postsynaptic current. Xenon selectively inhibits the NMDA receptor-mediated component of the current but has little effect on the AMPA/kainate receptor-mediated component. CONCLUSIONS: For both isoflurane and xenon, the most important targets appear to be postsynaptic. The authors' results show that isoflurane and xenon have very different effects on GABAergic and glutamatergic synaptic transmission, and this may account for their differing pharmacologic profiles.     

Mechanism of the endothelium-dependent vasodilator effect of an alcohol-free extract obtained from a vinifera grape skin.

An alcohol-free grape-skin extract (GSE) obtained from skins of Vitis labrusca has significant anti-hypertensive, antioxidant and vasodilator effects. According to our previous results, the vasodilator effect of GSE in the isolated mesenteric vascular bed (MVB) of the rat is dependent on endothelium and partially dependent on nitric oxide (NO). In the MVB of the rat pre-contracted with norepinephrine (NE), bolus injections of GSE induced a long-lasting dose-dependent vasodilation that is significantly reduced after the treatment with 1H-[1,2,3] oxadiazolo [4,4-a] quinoxalin-1-one (ODQ). Additionally, in vessels pre-contracted with norepinephrine and depolarized with KCl (25 mM) or treated with Ca(2+)-dependent K(+)-channel blockers charybdotoxin (ChTx) plus apamin, the vasodilator effect of GSE was significantly reduced and almost abolished by ChTx plus apamin plus L-NAME. However, the vasodilator effect of GSE was unaffected by D-Arg[Hyp(3),Thi(5),D-Tic(7),Oic(8)]bradykinin (HOE-140), atropine, yohimbine, pyrilamine and 4-aminopyridine (4-AP). The vasoconstriction response elicited by bolus injection of KCl was not affected by GSE, whereas the vasoconstrictor response induced by NE was dose-dependently and completed inhibited by GSE in the presence but not in the absence of endothelium. However, NE-induced vasoconstriction in calcium-free condition or without endothelium was not reduced by GSE. The present results demonstrate that GSE induces a vasodilator effect in the rat MVB, which is dependent on NO in combination with endothelium-derived hyperpolarizing factor (EDHF). Additionally, our results indicated that extracellular Ca(2+) has an important role on the endothelium-dependent vasodilator effect induced by GSE.     

Clinical and biological aspects of sentinel node biopsy in malignant melanoma — an update

The diagnostic usefulness of sentinel lymph node biopsy (SLNB) has been well established, but its therapeutic value remains unproven. First introduced by Morton and colleagues, the SLNB procedure is now widely available, and markedly enhances our ability to pathologically stage the regional nodes. Although the SLN status is acknowledged as the most powerful indicator of prognosis in melanoma, there is no evidence to-date, of survival advantage for complete lymphadenectomy in SLN-positive patients. Also, there is no effective adjuvant therapy that could benefit these sentinel node-positive patients, as yet. Additionally, new data have emerged indicating a possible increase in local/in-transit recurrence following complete lymphadenectomy in sentinel node-positive patients. To understand fully and to evaluate these observations we need information from randomized controlled trials. Major changes have occurred following the latest revision of melanoma staging system (AJCC, 6th edition). Concerning N category, these include the incorporation of the number of metastatic lymph nodes, the tumour burden of nodal metastases, and the ulceration of the primary tumour. The data obtained from the new staging system will reflect differences in prognosis that were not previously emphasized and which, we hope, will serve as a guide to more accurate analysis of metastatic pathways in cutaneous melanoma as well as a rationale for new forms of treatment.