Paul Urso

Human acetylator genotype: Relationship to colorectal cancer incidence and arylamine N-acetyltransferase expression in colon cytosol

Polymorphic expression of arylamine N-acetyltransferase (EC 2.3.1.5) may be a differential risk factor in metabolic activation of arylamine carcinogens and susceptibility to cancers related to arylamine exposures. Human epidemiological studies suggest that rapid acetylator phenotype may be associated with higher incidences of colorectal cancer. We used restriction fragment length polymorphism analysis to determine acetylator genotypes of 44 subjects with colorectal cancer and 28 non-cancer subjects of similar ethnic background (i.e., approximately 25% Black and 75% White). The polymorphic N-acetyltransferase gene (NAT2) was amplified by the polymerase chain reaction from DNA templates derived from human colons of colorectal and non-cancer subjects. No significant differences inNAT2 allelic frequencies (i.e., WT, M1, M2, M3 alleles) or in acetylator genotypes were found between the colorectal cancer and non-cancer groups. No significant differences inNAT2 allelic frequencies were observed between Whites and Blacks or between males and females. Cytosolic preparations from the human colons were tested for expression of arylamine N-acetyltransferase activity. Although N-acetyltransferase activity was expressed for each of the arylamines tested (i.e., p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, β-naphthylamine), no correlation was observed between acetylator genotype and expression of human colon arylamine N-acetyltransferase activity. Similarly, no correlation was observed between subject age and expression of human colon arylamine N-acetyltransferase activity. These results suggest that arylamine N-acetyltransferase activity expressed in human colon is catalyzed predominantly by NAT1, an arylamine N-acetyltransferase that is not regulated byNAT2 acetylator genotype. The ability to determine acetylator genotype from DNA derived from human surgical samples should facilitate further epidemiological studies to assess the role of acetylator genotype in various cancers.

Alterations in the humoral immune response and tumor frequencies in mice exposed to benzo[a]pyrene and x‐rays before or after birth

Pregnant mice were given one of three treatment regimens during mid (11 to 13 d) or late (16 to 18 d) gestation: benzo[a]pyrene (BaP) (150 μg/g body weight), 150 R X‐irradiation, or X‐irradiation + BaP. A group of virgin mice were injected with BaP after birth (at 1 wk or 16 wk) with doses similar to or higher than those to which progeny of pregnant mice were exposed in utero. The offspring exposed during fetal life showed a marked suppression in anti‐SRBC plaque‐forming cells (PFC) 1 wk after birth (pups), followed by an apparent recovery at 4 wk. Return to the immuno‐suppressed state seen in pups was most noticeable in adults encountering BaP alone during gestation. In this group, increased tumor incidence was associated with a sustained reduction in PFC response. In contrast, after fetal exposure to X‐rays or injection with BaP postnatally, immune competence was not appreciably different from normal in late life and tumor incidence was only slightly higher than controls with X‐rays or a high dose (150 ...

QUANTITATIVE AND FUNCTIONAL CHANGE IN T CELLS OF PRIMIPAROUS MICE FOLLOWING INJECTION OF BENZO(a)PYRENE AT THE SECOND TRIMESTER OF PREGNANCY

Progeny from benzo(a)pyrene (BP) exposed (150 micrograms/g body weight) primiparous mothers, injected during the second trimester of pregnancy, are severely compromised immunologically. In view of maternal-fetal associations, we studied, during pregnancy and postpartum, the quantitative and functional status of the maternal T cell population in the thymus and/or spleen. In the thymus, there is an exacerbated depression in the amounts of thymocytes, (theta +, Lyt 1+, Lyt 2+ cells) during pregnancy relative to the corn oil-injected controls, which is sustained postpartum. In the spleen, while there are inconsistencies in the level of theta + cells, The Lyt 1+ are depressed postpartum relative to virgins, but the Lyt 2+ are enhanced during pregnancy and postpartum, reaching levels greater than 700-fold of controls. In controls, while the number of Lyt 1+ cells was higher than BP-exposed mice or virgins during pregnancy, they virtually disappeared postpartum. A similar but reversed image is mirrored by the Lyt 2+ cells, i.e., they were virtually absent during pregnancy but increased postpartum. Splenic allogeneic and syngeneic mixed lymphocyte responses were subnormal. The data show that BP disrupts the maternal T cell repertoire, leading to an accumulation of Lyt 1-2+ cells, and suggest that splenic disruption may be due to changes in the differentiation potential of T precursor cells. These changes not only are most likely to affect maternal defenses, but also may have a direct bearing on the establishment of progeny immune status.

Suppression of T Lymphocyte Proliferation to Antigenic and Mitogenic Stimuli by Benzo(α)pyrene and 2-Aminofluorene Metabolites

Here, we attempt to reveal how 2-aminofluorene (AF), benzo(α)pyrene (BP) and their major metabolites affect T–cell responses to antigenic and mitogenic stimuli. P-450-related metabolism of these parent compounds to metabolites seems to precede the observed immunosuppression; therefore, we investigated the influence of α-naphthoflavone (P-450 inhibitor) and β-naphthoflavone (P-450 inducer) on BP and AF immunosuppression. We used proliferative responses to concanavalin A and the allogeneic mixed lymphocyte response as correlates of immunosuppression. We also attempted to correlate DNA-adduction to the extent of observed immunosuppression for AF and BP metabolites. These data show that the pathway to the strongest immunosuppressive agents for polycyclic aromatic hydrocarbons and arylamines are divergent and related to metabolism by P450 enzymes.

Variation in the response of T cells to Concanavalin A after in vitro exposure to benzo[A]pyrene and 2-aminofluorene

The ability of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BP) and its metabolites to be immunosuppressive has been well documented by many investigators. The arylamine, 2-aminofluorene (AF) and its metabolic intermediates have not been as widely studied in this regard. Here, we investigate the effect of BP, 3-hydroxy-BP (3-OH-BP), AF, N-hydroxy-AF (N-OH-AF) and acetyl-AF (AAF) on T-cell proliferation using the T-cell mitogen, Concanavalin A (ConA). These compounds as well as BP-7, 8-diol-9, 10-epoxide (BPDE) were also used to determine their effect on T-cell-mitogen binding. Both AF and BP are substrates for the P-450 and flavin-containing monooxygenase enzyme system, which can be induced with beta-naphthoflavone (beta NF). We incubated beta nF with BP and AF to determine the effect of a P-450 inducer on BP and AF mediated-ConA suppression. Here we demonstrate that BP, 3-OH-BP, AF, and AAF are able to suppress the proliferative response to ConA, while N-OH-AF cannot. Further, we show that BP, 3-OH-BP, BPDE, AF and N-OH-AF do not alter the ability of ConA to bind the mitogen receptor of splenic T-cells, indicating an intracellular mechanism for suppression. Studies with beta NF indicate that this P-450 inducer enhances the anti-proliferative effect of BP, while it abolishes this effect of AF.

Immunomodulation in progeny from thymectomized primiparous mice exposed to benzo(a)pyrene during mid-pregnancy.

Previous studies have shown that Benzo(a)pyrene (B(a)P3) given to non-thymectomized (NTX) female mice alters expression of T cell subsets and suppresses cell mediated immunity (CMI) and humoral immunity (HI) in the progeny. Thus, maternal exposure to B(a)P may influence changes in progeny immune status. To understand how maternal cellular and humoral factors influence embryonic development of progeny immunity, adult female mice were thymectomized (TX) at 6 weeks, mated and injected with 150 μg B(a)P)/g body weight at 12 days of pregnancy. After B(a)P exposure, the following studies were performed: (A) Maternal reproductive capacity and survival rate of progeny; (B) Detection of T cells in progeny thymus; (C) Functional characteristics of progeny thymus or spleen. Maternal thymectomy and B(a)P exposure reduced average litter size by 40%. Serological sensitivity of thymus cells with anti-Thyl + complement occurred at a higher dilution of mAb in progeny from TX mothers exposed to B(a)P, suggesting that B(a)P-thymectomy led to increased sensitivity of developing thymocytes to mAb plus complement. Progeny from TX mothers exposed to B(a)P showed enhanced thymic CMI, but suppressed splenic CMI and HI. Thus, thymectomy prevents CMI immunosuppression by B(a)P, while HI is still suppressed. These results indicate that the maternal thymus is necessary for incurring the effect of B(a)P on progeny CMI.

Murine Fetal Liver Augments Proliferation in an Allogenic Mixed Lymphocyte Culture: Benzo(A)Pyrene Reduces Augmentation

The effect of cells from the liver of C3H fetuses syngeneic to splenic responder cells on an allogeneic mixed lymphocyte response (MLR) was studied. After fractionation on ficoll-hypaque, interface cells from corn oil (vehicle for BP) or normal fetal liver (FL) controls (CO), obtained from 17-19 days gestation, enhanced proliferation in the mixed lymphocyte culture (> 2-fold), while cells from FLs transplacentally exposed to benzo(a)pyrene (BP) showed a decreased capacity for augmentation (> 2-fold less than CO). Unfractionated CO-FL cells at 0.5 x 10(6) did not augment proliferation, but at 0.25 x 10(6) enhancement with control FL cells was significantly higher than with BP-FL cells. Pelleted cells from BP- and CO-FLs were severely suppressive at the higher dose, while at 0.25 x 10(6) proliferation was augmented with CO-FL cells, but not affected with BP-FL cells. At doses of 0.1 x 10(6) or less, no effect was observed for either control or BP-FL cells. These data indicate that: a) FL cells syngeneic to responder cells of an allogenic mixed lymphocyte culture (MLC) have an augmenting but not suppressive capacity on cell proliferation; b) in utero insult with BP modifies the capacity of FL cells to augment proliferation in the MLC.

Changes in Peripheral Blood Cells in Mice After Injection with Benzo(A)Pyrene During Pregnancy

AbstractPregnant C3H/Anfcum mice were injected ip with 150 ug benzo-(a)pyrene (BP)/g body weight at the second trimester (12 days). Quantitative and differential changes were assayed in the peripheral blood leukocytes and erythrocytes at various times before and after mating and treatment. Within 5 days after injection, a 2- to 4-fold reduction in leukocytes was observed when compared to controls [corn oil (vehicle for BP)-injected pregnant females] which persisted into the 10th postpartum day. The erythrocytes were also significantly reduced but not to the same degree (1.2- to 1.5-fold). Depression in white blood cells is attributed to lymphocyte depletion since the granulocytes were virtually unchanged and the lymphocyte to granulocyte ratio, ordinarily >2 was 1 or < one. No change in monocytes was observed and none of the cell populations, including the erythrocytes, appeared to be abnormal (e.g., no increase in reticulocytes). A moderate reduction (1.5-fold) in erythrocytes and leukocytes also occurre...

Alterations in CD4+, CD8+, Vγ3, Vγδ, and/or Vα βT-Lymphocyte Expression in Lymphoid Tissues of Progeny After In Utero Exposure to Benzo(α)pyrene

That benzo〈 α)pyrene (Bα P) decreases both humoral and cell-mediated immunity, and leads to increases in progeny tumor development after in utero insult, suggests that T- and B-lymphocytes are made defective in exposed offspring. In the study here, C3H mice were injected once with Bα P (150 μ g/g BW) at day 12 of pregnancy and progeny lymphoid tissues were excised during gestation (day 18; GD18) or at 1 or 6 weeks post-partum. The isolated lymphoid cells were analyzed by flow cytometry/immunofluorescence or assessed for function. In Bα P-exposed fetuses, thymic Thy1+ cell levels were decreased (relative to levels in organs of corn oil-exposed dam progeny). In addition, for up to 6 weeks post-birth, CD4+CD8+ (double positive; DP) cells were virtually absent and levels of CD4−CD8− (double negative; DN) cells were consistently at ε 90%. With regard to single positive (SP) cells, CD4+ cell levels were also decreased in tissues at GD18 up through 6 weeks post-birth; CD8+ cell levels were increased, but only in pups at 1-week and 6-weeks post-birth. In 1-week-old progeny, spleen CD8+ cell levels were quantitatively unchanged, though CD4+ levels were reduced 2–4-fold and CD4−CD8− DN levels significantly increased. With respect to TCRs, fetal levels of thymic CD3Vγ3+ and CD3Vγ δ+ cells were decreased; levels of CD3Vαβ cells were only slightly depressed. The latter results contrast sharply with a strong reduction in CD3Vαβ cells in the fetal livers of Bα P-exposed progeny. Interestingly, these livers also strongly evidenced a presence of BαP-7,8-dihydrodiol-9,10-epoxide metabolite. When assessed for any change in function, the CD4+, Thy1+ cells isolated from Bα P-exposed progeny tissues responded weakly (relative to controls) to ConA and in an allogeneic MLR. Taken in totality, the results here strengthen our original hypothesis that BαP can create a favorable milieu for tumor growth progression in progeny of exposed mothers by affecting development of sufficient numbers of functional lymphocytes in the offspring.

Changes in biologic and/or immunologic parameters induced by intratracheal instillation of pregnant mice with benzo(α)pyrene are potentially confounded by anesthesia

Benzo(α)pyrene (B(α)P) is a potent multiorgan carcinogen released into the atmosphere from commercial, domestic, and industrial sources. Studies using animal models have shown that giving B(α)P parenterally to pregnant animals (i.e., dams) led to increased tumor frequency and sensitivity to tumorigenesis in their progeny. The authors’ studies also showed that the progeny of the B(α)P-exposed dams displayed increased deficiencies in cell-mediated and humoral immune functions, changes among T-cell subsets in developing lymphoid tissues, and significant expression of B(α)P-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in thymic, splenic, and (fetal) liver tissues. The authors evaluated whether similar biologic/immunologic effects of B(α)P seen earlier in parenterally exposed mouse dams (and offspring) occurred if dams were exposed to B(α)P via the lungs. Pregnant dams were subjected to intratracheal instillation of B(α)P (at 1 mg/ml corn oil, 0.1 ml/instillate) beginning on day 11 of pregnancy (GD 11) and again on GDs 12 and 14. In each case, the dams were anesthetized with metofane. Other dams were left untreated (controls), anesthetized only, or anesthetized and then instilled with vehicle. Effects of the B(α)P exposures included lower dam body weights during gestation, decreased postbirth pup survival, increased pup tumor frequency, and decreased mixed-lymphocyte responses by pup lymphocytes. These studies also revealed that metofane imparted effects on the dams and progeny. These effects equaled the B(α)P treatments alone; in other instances, the metofane had no impact, and thus questions the observed biologic/immunologic effects of B(α)P induced in pregnant mice (and their progeny), which might have been confounded by use of this (or potentially other) anesthesia.