Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.     

Metabolic activation and carcinogen-DNA adduct detection in human larynx

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.     

Beta-glucuronidase and hyperbilirubinemia in breast-Fed babies

In the present study we analyzed breast milk samples from mothers of breast-fed, healthy, term babies with unexplained prolonged jaundice for beta-glucuronidase activity. The differences of enzyme activity were not statistically significant in comparison to a control group of nonjaundiced newborns. We conclude that beta-glucuronidase is not the only cause of prolonged jaundice in neonates.     

Effect of the genetic background on the reproduction of leptin-deficient obese mice

Obesity is often associated with an impairment of the hypothalamic-pituitary-gonadal axis. The leptin-deficient ob/ob mouse model is characterized by a morbid obesity with a sterility in males and females that is corrected by continuous leptin treatment. Since ob/ob mice are maintained on the C57BL/6J inbred genetic background, we sought to determine whether their infertility can be corrected without leptin treatment but via the effect of modifier genes brought into the obese-sterile phenotype by a different genetic background. Thus, we generated via an F2 intercross ob/ob mice on a mixed C57BL/6J-BALB/cJ genetic background and assayed them for fertility by mating with wild-type C57BL/6J mice. Whereas genetically heterogeneous F2 obese females remained sterile like male and female C57BL/6J ob/ob mice, 41% of F2 C57BL/6J-BALB/cJ obese males were capable of reproducing despite a morbidly obese state. Therefore, the sterility of the original C57BL/6J ob/ob mouse model was genetically corrected independently of its obese state via the effects of modifier genes. Unlike testosterone levels, triglyceride levels, and testes weight-to-body weight ratios, which were all higher in fertile vs. sterile mice, glucose levels were similar in both groups, indicating that the underlying hyperglycemia of ob/ob mice was not an impediment to the onset of fertility. A genome-wide scan in F2 ob/ob males resulted in the localization of four modifier loci on chromosomes 1, 3, 5, and 14 with respective quantitative traits consisting of number of pregnancies, testes weights normalized to body weights, body weight at 8 weeks of age, and circulating testosterone. We conclude that the inheritance of modifier genes at the identified loci acts to promote fertility of otherwise sterile leptin-deficient obese male mice.     

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.     

The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcgamma binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.     

Intra-individual comparison of 3(R)-BMIPP and 3(S)-BMIPP isomers in humans

The racemic 15-(p-iodophenyl)-3(R,S)-methylpentadecanoic acid (BMIPP) is currently used at several centers for myocardial metabolic imaging with SPECT. Recently, the 3(R)-BMIPP isomer showed a 20%-25% higher myocardial uptake and lower liver uptake than 3(S)-BMIPP in fasted rats. The aim of this study was to determine if these differences in myocardial and liver uptake also occur in humans. METHODS: Iodine-123-labeled 3(R)-BMIPP and 3(S)-BMIPP isomers were injected at rest, on two separate days, in six patients with stable coronary artery disease. Dual-head, whole-body scintigraphy was performed 20 min and 3 hr after injection. SPECT cardiac imaging was performed 60 min after injection. RESULTS: Myocardial activity averaged (% injected dose +/- s.d.) 3.15 +/- 0.49 versus 3.01 +/- 0.44 at 20 min (p = ns) and 2.64 +/- 0.38 versus 2.55 +/- 0.41 at 3 hr postinjection (p = ns) for the 3(R)-BMIPP and 3(S)-BMIPP isomers, respectively. Liver activity averaged 9.50 +/- 1.18 versus 9.44 +/- 0.66 at 20 min and 5.33 +/- 0.64 versus 5.43 +/- 0.66 at 3 hr, respectively (p = ns). SPECT showed no difference in the distribution of the two isomers between normal and infarcted myocardium. CONCLUSION: There is no significant difference in myocardial and liver distribution of the 3(R)-BMIPP and 3(S)-BMIPP isomers in humans.