Phalloplasty Complicated by Penile Artery Thrombosis, Recurrent Extended-Spectrum Beta-Lactamase (ESBL) Urinary Tract Infection (UTI), Colovesical Fistula, and Enterococcus Faecalis Endocarditis.

Enterococcus faecalis is an enteric microorganism that, if introduced into the vasculature, is an uncommon cause of infective endocarditis. Timely diagnosis, appropriate treatment measures, and close follow-up are key to therapeutic success. Antibiotic therapy is the mainstay of therapy, and surgical intervention is sometimes indicated. Here we present a novel case of a 45-year-old transgender male with Factor V Leiden deficiency who was found to have Enterococcus faecalis mitral valve endocarditis due to the postoperative complications of colovesical fistula formation leading to extended-spectrum beta-lactamase (ESBL) urinary tract infection (UTI) and E. faecalis bacteremia in the setting of recent phalloplasty, mastectomy, and vaginal eversion.

Effects of subchronic acrylamide treatment on the endocrine pancreas of juvenile male Wistar rats.

Acrylamide (AA) is a well-known industrial monomer with carcinogenic, mutagenic, neurotoxic and endocrine disruptive effects on living organisms. AA has been the subject of renewed interest owing to its presence in various food products. We investigated the potential adverse effects of oral AA treatment on the endocrine pancreas of juvenile rats using histochemical, immunohistochemical, stereological and biochemical methods. Thirty juvenile male Wistar rats were divided into one control and two AA treatment groups: one treated with 25 mg/kg AA and the other treated with 50 mg/kg AA for 21 days. We found a significant decrease in ß-cell mass. The significant decrease in ß-cell optical density and unchanged blood glucose levels indicate that normoglycemia in AA treated rats may result from intensive exocytosis of insulin-containing secretory granules. By contrast with ß-cells, we observed increased α-cell mass. The slight increase in α-cell cytoplasmic volume suggests retention of glucagon in α-cells, which is consistent with the significant increase in α-cell optical density for AA treated animals. The number of islets of Langerhans did not change significantly in AA treated groups. Our findings suggest that AA treatment causes decreased ß-cell mass and moderate α-cell mass increase in the islets of Langerhans of juvenile male Wistar rats.

Diagnosis and Treatment of Venous Malformations. Consensus Document of the International Union of Phlebology (IUP): updated 2013.

Venous malformations (VMs) are the most common vascular developmental anomalies (birth defects) . These defects are caused by developmental arrest of the venous system during various stages of embryogenesis. VMs remain a difficult diagnostic and therapeutic challenge due to the wide range of clinical presentations, unpredictable clinical course, erratic response to the treatment with high recurrence/ persistence rates, high morbidity following non-specific conventional treatment, and confusing terminology. The Consensus Panel reviewed the recent scientific literature up to the year 2013 to update a previous IUP Consensus (2009) on the same subject. ISSVA Classification with special merits for the differentiation between the congenital vascular malformation (CVM) and vascular tumors was reinforced with an additional review on syndrome-based classification. A "modified" Hamburg classification was adopted to emphasize the importance of extratruncular vs. truncular sub-types of VMs. This incorporated the embryological ongm, morphological differences, unique characteristics, prognosis and recurrence rates of VMs based on this embryological classification. The definition and classification of VMs were strengthened with the addition of angiographic data that determines the hemodynamic characteristics, the anatomical pattern of draining veins and hence the risk of complication following sclerotherapy. The hemolymphatic malformations, a combined condition incorporating LMs and other CVMs, were illustrated as a separate topic to differentiate from isolated VMs and to rectify the existing confusion with name-based eponyms such as Klippei-Trenaunay syndrome. Contemporary concepts on VMs were updated with new data including genetic findings linked to the etiology of CVMs and chronic cerebrospinal venous insufficiency. Besides, newly established information on coagulopathy including the role of D-Dimer was thoroughly reviewed to provide guidelines on investigations and anticoagulation therapy in the management of VMs. Congenital vascular bone syndrome resulting in angio-osteo-hyper/hypotrophy and (lateral) marginal vein was separately reviewed. Background data on arterio-venous malformations was included to differentiate this anomaly from syndromebased VMs. For the treatment, a new section on laser therapy and also a practical guideline for follow up assessment were added to strengthen the management principle of the multidisciplinary approach. All other therapeutic modalities were thoroughly updated to accommodate a changing concept through the years.

Vitamin C Depletion in Prenatal Guinea Pigs as a Model of Lissencephaly Type II.

Humans and guinea pigs are unable to produce vitamin C, with deficiency resulting in a well-known disorder of collagen synthesis. Pial basement membrane structure preservation is essential in the proper migration of neurons. In our study, intrauterine deprivation of vitamin C in guinea pig fetuses led to a collagen synthesis disorder, weakness, and finally a breach of pial basement membrane. We found excessive migration of the external germinal layer cells into the subarachnoid space of the cerebellum through defects in the pial basement membrane. The changes ranged from focal rupture of pial basement membranes to their complete disintegration. The loss of proper folia formation resulted in macroscopically visible flattening of the cerebellar surface. Different grades of dysplastic changes in the folia of the cerebellar cortex were observed in 2 experimental groups assigned different limits to mark the time of commencement and duration of vitamin C deprivation. The most severe form of dysplastic changes was characterized by marked irregularity of the cerebellar cortex similar to that in lissencephaly type II. Thus, prenatal vitamin C deficiency represents a novel animal model to study the effects of collagen synthesis on development of breaches in the pial basement membrane, disordered migration of neurons, dysplasia of cerebellar cortex, and the pathogenesis of lissencephaly.

Guideline. Diagnosis and treatment of venous malformations. consensus document of the international union of phlebology (iup): updated-2013.

Venous malformations (VMs) are the most common vascular developmental anomalies (birth defects). These defects are caused by developmental arrest of the venous system during various stages of embryogenesis. VMs remain a difficult diagnostic and therapeutic challenge due to the wide range of clinical presentations, unpredictable clinical course, erratic response to the treatment with high recurrence/persistence rates, high morbidity following nonspecific conventional treatment, and confusing terminology. The Consensus Panel reviewed the recent scientific literature up to the year 2013 to update a previous IUP Consensus (2009) on the same subject. ISSVA Classification with special merits for the differentiation between the congenital vascular malformation (CVM) and vascular tumors was reinforced with an additional review on syndrome-based classification. A "modified" Hamburg classification was adopted to emphasize the importance of extratruncular vs. truncular subtypes of VMs. This incorporated the embryological origin, morphological differences, unique characteristics, prognosis and recurrence rates of VMs based on this embryological classification. The definition and classification of VMs were strengthened with the addition of angiographic data that determines the hemodynamic characteristics, the anatomical pattern of draining veins and hence the risk of complication following sclerotherapy. The hemolymphatic malformations, a combined condition incorporating LMs and other CVMs, were illustratedas a separate topic to differentiate from isolated VMs and to rectify the existing confusion with namebased eponyms such as Klippel-Trenaunay syndrome. Contemporary concepts on VMs were updated with new data including genetic findings linked to the etiology of CVMs and chronic cerebrospinal venous insufficiency. Besides, newly established information on coagulopathy including the role of D-Dimer was thoroughly reviewed to provide guidelines on investigations and anticoagulation therapy in the management of VMs. Congenital vascular bone syndrome resulting in angio-osteo-hyper/hypotrophy and (lateral) marginal vein was separately reviewed. Background data on arterio-venous malformations was included to differentiate this anomaly from syndrome-based VMs. For the treatment, a new section on laser therapy and also a practical guideline for follow up assessment were added to strengthen the management principle of the multidisciplinary approach. All other therapeutic modalities were thoroughly updated to accommodate a changing concept through the years.

Contribution of immunohistochemistry in the differential diagnosis of non-small cell lung carcinomas on small biopsy samples.

PURPOSE: Targeted therapy increases survival and the quality of life of non-small cell lung cancer (NSCLC) patients but it needs precise histological subtyping. The present study evaluated 6 monoclonal antibodies for the differential diagnosis of NSCLC on small-sized tissue samples. METHODS: 50 small-sized tissue samples were obtained by bronchoscopy or fine needle aspiration biopsy (FNAB). According to morphology before immunohistochemistry 2 squamous cell carcinomas (SCC), 6 adenocarcinomas (AC), 9 NSCLC-probably SCC, 11 NSCLC-probably AC and 22 unclassified NSCLCs were diagnosed. Thyroid transcription factor-1 (TTF-1), cytokeratin 5/6, cytokeratin 7, p63, and the neuroendocrine markers CD56 and synaptophysin were used in the differential diagnosis of NSCLC. RESULTS: After immunohistochemistry 13 (26.0%) SCC, 27 (54.0%) AC, 3 (6.0%) NSCLC with neuroendocrine differentiation (NSCLC-NE) and 7 (14.0%) NSCLC- unclassified were diagnosed. Twenty-two NSCLC- unclassified were further diagnosed as SCC (n=7), AC (n=7) NSCLC-NE (n=2) and 6 remained NSCLC- unclassified. Significant difference was found between definitely diagnosed 8 NSCLCs and 15 ACs (20.5 vs. 38.57percnt;, p=0.008). TTF-1 and cytokeratin 7 were expressed in 85.2% (23/27) of AC, and cytokeratin 5/6 and p63 in 100% (13/13) of SCC. Positivity of CD56 and synaptophysin in 3 NSCLC determined NSCLC-NE. CONCLUSION: No one monoclonal antibody is totally specified for one histological type of tumor and its origin. Combination of TTF-1, cytokeratin 7, p63, cytokeratin 5/6, CD56 and synaptophysin allows for differentiation of NSCLC but Napsin-A for AC differentiation and chromogranin A for NSCLC-NE differentiation should be added in an optimal panel.

The impact of heroin on visual memory.

INTRODUCTION AND AIM: Heroin abuse can lead to organic damage of cerebral structures, including sequels in cognitive and affective sphere, which are in positive relation with the duration of substance usage. Memory is one of the cognitive functions which is highly sensitive to opiate toxic effects. The aim of this research was determination of heroin impact on the visual memory of addicts, as well as the existence of specific relation of potential deficiencies in visual memory with the duration of substance use. METHODS: The research included 90 examinees, divided into three groups, depending on the duration of heroin intake. We used questionnaire for basic socio-demographic and addictological traits of examinees; Wechsler's scale for the assessment of the intelligence and Visual Memory Test (TVP), for the assessment of the visual memory. RESULTS: The achievements of heroin addicts with different duration of the substance abuse differ significantly (F = 1.83; df = 12; p < 0.05). Total number of errors examinees make in the first series of TVP (immediate visual memory) grows, almost linearly in the function of the duration of heroin abuse (p < 0.05), but in neither of groups meets criteria for the visual memory impairment. Deficiency of the delayed visual memory occurs in examinees who use heroin for one (total number of errors = 6.46; participation of typical organic errors = 31.7%) and longer than five years (total number of errors = 7.66; participation of typical organic errors = 26.7%). Univariate covariance analysis separates the average daily dosage of heroin as the most significant variable that contributes to the expression of the aforementioned deficiencies (F = 4.21; df = 2; p < 0.05). CONCLUSION: Heroin abuse leads to damage of delayed visual memory, whereby for the observed effect intake of the substance for a period longer than one year is necessary.

Gender and age trends of histological types of lung cancer in a 20-year period: pathological perspective.

PURPOSE: To find out the trends of distribution in different histological types of lung cancer in both genders in a period of 20 years. METHODS: The most frequent histological types of lung cancer in tissue specimens obtained by bronchoscopy or percutaneous needle biopsy were analysed in terms of age and gender. The studied population included 6289 patients (16.6% females and 83.1% males). Statistical significance was established by x(2) test at the level p<0.05. RESULTS: Squamous cell carcinoma (SCC) prevailed in the total number of patients in all investigated years (58.0%), and separately in male (60.4%) and female (45.7%) patients. This histological type was predominant in all age groups in both genders (41.6% in males and 38.1% in females). CONCLUSION: SCC has the highest incidence in Serbia. Continuous campaign against smoking and helping its cessation, improving working and socioeconomic conditions is a strategy for decreasing all histological types of lung cancer patients.

Prediction and biochemical characterization of intrinsic disorder in the structure of proteolysis-inducing factor/dermcidin.

Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single approximately 16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired alpha-helix and beta-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR, DisEMBL, DisProt, and GlobPlot computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.

Prediction and biochemical characterization of intrinsic disorder in the structure of proteolysis-inducing factor/dermcidin

Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single ~16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired á-helix and â-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR®, DisEMBL™, DisProt, and GlobPlot™ computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.

Expression and prognostic significance of Cathepsin L in early cutaneous malignant melanoma.

Cathepsins are papain-like lysosome cysteine proteases involved in tumor growth, invasiveness and spread, angiogenesis and alteration in immune and inflammatory responses. We investigated the differences in cathepsin L (CatL) concentrations in primary cutaneous malignant melanoma stage I and normal skin and correlated these values with well-established malignant melanoma prognostic factors. The study was performed on 36 patients (17 men and 19 women; mean age 54 years; range 21-84 years) with histological confirmed primary malignant melanomas less than 1.5 mm thick. The CatL concentrations were measured in 36 pairs of triton extracts of cytosols prepared from the tumor and adjacent normal tissue samples (matched pairs). The CatL concentrations were determined by commercially available enzyme-liked immunosorbent (ELISA) assay from KRKA (Novo Mesto, Slovenia). Significantly higher concentrations of CatL were detected in malignant melanomas than in normal surrounding skin (6.73 vs. 1.42 ng/mg total protein (mgp), p<0.001). Significant correlations between malignant melanoma and normal skin concentrations for CatL were found. The malignant melanoma CatL concentrations correlated significantly with normal skin (r=0.38; p=0.021). CatL concentrations were significantly lower (p<0.01) in the malignant melanomas of Breslow thickness 0.75 mm, Clark invasion of >or=II and

Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs.

Exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin resulted in the appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin (96.2-fold) and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin. Slight cross-resistance to two MDR-unrelated drugs 8-Cl-cAMP and sulfinosine was observed. Flow cytometry analysis showed that the accumulation of doxorubicin in the resistant cells was 88.4% lower than in the parental cells. Also, verapamil significantly decreased the efflux rate in NCI-H460 and NCI-H460/R cells, whereas curcumin inhibited the efflux in NCI-H460 cells only. Gene expression data confirmed the induction of mdr1 (P-gp), as judged by the observed 15-fold increase in its mRNA concentration in doxorubicin-resistant NCI-H460/R cells. In contrast, mrp1 and lrp expression was unaffected by the doxorubicin resistance. Further work should develop a rationale for a novel treatment of NSCLC with appropriate modulators of resistance aimed at improving the outcome of the acquired drug resistance.

Adenomyoma of the common hepatic duct.

BACKGROUND: Adenomyoma occurs most commonly in the fundus of the gallbladder, seldom in other parts of the gallbladder and rarely in the extrahepatic biliary tree, where most lesions are localised to the common bile duct or papilla of Vater. Adenomyoma of the common hepatic duct is extremely rare. To the best of our knowledge, only three cases have been reported so far. CASE OUTLINE: A 51-year-old woman was admitted with a three month history of attacks of right upper abdominal pain, nausea, vomiting and fever. Laboratory data, ultrasonography, ERCP and CT confirmed slight cholestasis and proximal bile duct dilatation due to a tumour within the common hepatic duct. Cholecystectomy was performed with excision of the suprapancreatic common bile duct including the convergence of the hepatic ducts plus lymphadenectomy and Roux-en-Y hepaticojejunostomy. Frozen section histology showed the benign nature of the lesion and a tumour-free resection line. Final histology showed adenomyoma. The patient has remained symptom free for more than 30 months. DISCUSSION: Although adenomyoma is a benign lesion and the surgical strategy has not been established, complete excision with frozen section is recommended to exclude small malignant foci and local recurrence as well as to avoid surgical over-treatment.

Oxidative damage and stress response from ochratoxin a exposure in rats.

Ochratoxin A (OTA) is a mycotoxin found in some cereal and grain products. It is a potent renal carcinogen in male rats, although its mode of carcinogenic action is not known. Oxidative stress may play a role in OTA-induced toxicity and carcinogenicity. In this study, we measured several chemical and biological markers that are associated with oxidative stress response to determine if this process is involved in OTA-mediated toxicity in rats. Treatment of male rats with OTA (up to 2 mg/ 24 h exposure) did not increase the formation of biomarkers of oxidative damage such as the lipid peroxidation marker malondialdehyde in rat plasma, kidney, and liver, or the DNA damage marker 8-oxo-7,8-dihydro-2' deoxyguanosine in kidney DNA. However, OTA treatment (1 mg/kg) did result in a 22% decrease in alpha-tocopherol plasma levels and a 5-fold increase in the expression of the oxidative stress responsive protein haem oxygenase-1, specifically in the kidney. The selective alteration of these latter two markers indicates that OTA does evoke oxidative stress, which may contribute at least in part to OTA renal toxicity and carcinogenicity in rats during long-term exposure.

Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid is a major detoxification pathway catalyzed by cytochrome P450 1A2.

Metabolic pathways of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) remain incompletely characterized in humans. In this study, the metabolism of MeIQx was investigated in primary human hepatocytes. Six metabolites were characterized by UV and mass spectroscopy. Novel metabolites were additionally characterized by 1H NMR spectroscopy. The carcinogenic metabolite, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline, which is formed by cytochrome P450 1A2 (P450 1A2), was found to be transformed into the N(2)-glucuronide conjugate, N(2)-(beta-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx), thought to be formed exclusively by the intestinal flora, were also identified. A novel metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), and it was the predominant metabolite formed in hepatocytes exposed to MeIQx at levels approaching human exposure. IQx-8-COOH formation is catalyzed by P450 1A2. This metabolite is a detoxication product and does not induce umuC gene expression in Salmonella typhimurium strain NM2009. IQx-8-COOH is also the principal oxidation product of MeIQx excreted in human urine [Turesky, R., et al. (1998) Chem. Res. Toxicol. 11, 217-225]. Thus, P450 1A2 is involved in both the metabolic activation and detoxication of this procarcinogen in humans. Analogous metabolism experiments were conducted with hepatocytes of untreated rats and rats pretreated with the P450 inducer 3-methylcholanthrene. Unlike human hepatocytes, the rat cell preparations did not produce IQx-8-COOH but catalyzed the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline as a major P450-mediated detoxication product. In conclusion, our results provide evidence of a novel MeIQx metabolism pathway in humans through P450 1A2-mediated C(8)-oxidation of MeIQx to form IQx-8-COOH. This biotransformation pathway has not been detected in experimental animal species. Considerable interspecies differences exist in the metabolism of MeIQx by P450s, which may affect the biological activity of this mutagen and must be considered when assessing human health risk.

Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes.

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.

Mutational analysis of the liver, colon and kidney of Big Blue rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline.

The heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) induces intestinal tumours and hepatocellular carcinomas in rats, but no tumourigenic effects have been identified in the kidney. The tissue-specific mutagenicity of IQ was studied at the lacI locus in the liver, colon and kidney of Big Blue transgenic rats. At the highest dosing regime of IQ (20 mg/kg for 5 consecutive days) the mean mutant frequencies were significantly increased above background (P < 0.05) and were highest in the liver (12.9 +/- 6.2 x 10(-5)), followed by colon (7.4 +/- 1.4 x 10(-5)) and kidney (5.9 +/- 0.8 x 10(-5)). The mutational spectra from the livers of IQ-treated rats was statistically significantly different to that from the livers of control rats (P < 0.01). The lacI mutation spectra of the liver, colon and kidney from IQ-treated rats were similar. These were characterized by an increase in GC transversions in the liver and colon and an increase in the proportion of 1 bp G:C deletions in the liver and kidney. A single G deletion in the sequence 5'-CGGGA-3', characteristic of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure, was detected in the liver and colon. A 2 bp GC deletion was identified at an identical position in the liver, colon and kidney. The colon was the only organ to contain two larger deletions of 13 and 33 bp. A preference was observed for base substitution mutations at guanine in the sequence 5'-CGC/T-3' and for 1 bp deletions at the guanine doublet in the sequence 5'-CGGA-3', especially in the liver and colon. Using the lacI gene as marker in the Big Blue rat model, the mutations identified in the IQ spectra have similarities to those identified for other HAAs studied in the same experimental system, but not to mutations identified in IQ-induced tumours.

Activation of heterocyclic aromatic amines by rat and human liver microsomes and by purified rat and human cytochrome P450 1A2.

The dietary mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are activated to genotoxins by rat and human liver cytochrome P450 (P450) 1A1- and 1A2-mediated N-oxidation. Immunoquantitation of 51 human liver samples revealed a wide range in P450 1A2 expression (10-250 pmol/mg of microsomal protein, median 71 pmol/mg), with 39% of the livers containing >100 pmol/mg of protein. There was no evidence for expression of P450 1A1 (<1 pmol/mg of protein). P450 1A2 levels were correlated to MeIQx and PhIP N-oxidation rates (r = 0.83, 0.73, respectively). In male Fischer-344 and Sprague-Dawley rats, hepatic P450 1A2 ranged from 5 to 35 pmol/mg of protein, while P450 1A1 was <1 pmol/mg. Animal pretreatment with 3-methylcholanthrene, beta-naphthoflavone, or polychlorinated biphenyls (PCB) resulted inasmuch as 340-fold and >1000-fold induction of P450 1A2 and 1A1, respectively, and a 220-fold increase in N-oxidation activity. Approximately 20% of the human samples were as active in N-oxidation and conversion of MeIQx to bacterial mutagens as microsomes of PCB-pretreated rats [3-4 nmol of NHOH-MeIQx formed min-1 (mg of protein)-1]. In contrast, microsomes from PCB-treated rats displayed higher rates of PhIP N-oxidation and activation to mutagens than the most active human liver microsomes [8-24 vs 2-4 nmol of HNOH-PhIP formed min-1 (mg of protein)-1]. Recombinant human P450 1A2 showed catalytic efficiencies of MeIQx and PhIP N-oxidation that were 10-19-fold higher than purified rat P450 1A2. Cytochrome P450 1A2 expression in rodent and human liver tissue varies greatly and there are considerable differences between the enzymes in the two species in the activation of some heterocyclic aromatic amines, which must be considered when assessing human health risk.

Formation and persistence of DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline in the rat and nonhuman primates.

The formation and persistence of the two principal DNA adducts of the food derived carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) have been investigated in rats and nonhuman primates. DNA adduct formation in the liver of male Fischer-344 rats occurred in a dose-dependent manner (0.01-20 mg/kg) where N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f] quinoline (dG-N2-IQ) accounted for approximately 60-80% and 20-40%, respectively, of the total adducts observed by 32P postlabeling. Similar DNA adduct profiles were observed in kidney and colorectal tissue of rats given a single oral dose of IQ (20 mg/kg) which, when given chronically to this species, results in tumorigenesis in liver and colorectum, but not in kidney. dG-C8-IQ was removed more rapidly than dG-N2-IQ from liver and kidney, but removal of both adducts from the colorectum closely followed cell replication. Similar DNA adduct profiles were observed in liver and extrahepatic tissues of nonhuman primates following a single dose of IQ (20 mg/kg). In chronically treated monkeys undergoing carcinogen bioassay, there was a sharp increase in the contribution of dG-N2-IQ to total DNA adducts in all slowly dividing tissues. There was no preferential accumulation of dG-N2-IQ in the colon, a tissue with a high rate of cell division, and dG-C8-IQ remained the predominant lesion. These findings point to a preferential removal of the dG-C8-IQ adduct by enzyme repair system(s) in slowly dividing tissues in both rats and nonhuman primates.