Induction of arylhydrocarbon hydroxylase and blast transformation in human blood lymphocytes

Variation in arylhydrocarbon hydroxylase(AHH) inducibility in human peripheral blood lymphocytes was studied. AHH inducibility and the degree of blast transformation were determined simultaneously using [³H]benzo(a)pyrene and [³H]thymidine, respectively. AHH inducibility in terms of induction ratio(induced level to basal) or induction ratio per unit of blast transformation varied at different culture time and at different phytohemagglutinin concentrations within the same individuals. However, the ratio of absolute induced AHH activity and unit of blast transformation gave persistent value for the same individuals, indicating that AHH inducibility of human lymphocytes should be expressed in this manner in the study of cancer susceptibility.     

Induction of aryl hydrocarbon hydroxylase in human lymphocytes and pulmonary alveolar macrophages--a comparison

Exposure of animals to cigarette smoke causes an increase in the levels of aryl hydrocarbon hydroxylase (AHH) in various tissues. The innate capacity for enzyme induction is genetically determined but the extent of induction and AHH levels in various tissues may vary. AHH levels in human pulmonary alveolar macrophages (PAMs) were determined and AHH inducibility of cultured lymphocytes from corresponding volunteers was determined. The inducibility by 3-methylcholanthrene of cultured lymphocytes was similar in both smokers and nonsmokers, ranging from 0.2 – 4.2 fold induction. AHH levels in nonsmoker PAMs was 0 – 0.020 units and in smokers was 0.032 – 0.253 units. The correlation of AHH activity in PAMs with lymphocyte inducibility was significant in both nonsmokers and smokers. The regression of AHH in PAMs as compared with AHH inducibility in lymphocytes was 8.5 fold higher in smokers than nonsmokers, reflecting the induction of AHH in PAMs by smoking.     

Aryl hydrocarbon hydroxylase in cultured lymphocytes of twins.

Measurement of aryl hydrocarbon hydroxylase (AHH) in cultured lymphocytes of 18 monozygotic and 30 dizygotic twin pairs showed that basal and induced AHH activity and AHH inducibility are heritable traits. The data are consistent with AHH inducibility being determined by a single or a very few polymorphic genes.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

Aryl hydrocarbon hydroxylase activity in pulmonary alveolar macrophages and lymphocytes from lung cancer and noncancer patients: A correlation with family histories of cancer

Aryl hydrocarbon hydroxylase (AHH) activity was measured in pulmonary alveolar macrophages (PAMs) and peripheral blood lymphocytes from cigarette smokers with and without primary lung cancer. Frequency distribution analysis of AHH induction ratios for the two groups revealed an increased number of individuals in the lung cancer patient group with high lymphocyte induction values (P less than 0.05). A similar increase was not shown for high-PAM AHH values in lung cancer patients (P greater than 0.2). When individual PAM and lymphocyte AHH values were compared between noncancer and lung cancer patients, a positive correlation was observed for noncancer patients (r=0.195, P less than 0.001), but no correlation of these values was noted for lung cancer patients. The lung cancer patients were divided into three subgroups of patients showing (I) high PAM and low lymphocyte AHH levels, (II) low PAM and low lymphocyte AHH levels, and (III) low PAM and high lymphocyte AHH levels. When the incidence of family history of cancer was compared for these subgroups, no family cancer history was recorded for persons in subgroup II; however, individuals in subgroups I and III presented family cancer history incidence of 9.5% and 39.3%, respectively. Patients in group III averaged 6 years younger than those in group I. These data suggest that familial factors may be identified among lung cancer patients and that these factors appear to associate as either a cause of an effect with the capacity of pulmonary alveolar macrophages and lymphocytes to be induced for AHH. The data support the hypothesis that high AHH values may be characteristic of lung cancer patients but show that enzyme values determined from a single tissue, either PAMs or lymphocytes, may not be appropriate for showing whether high AHH inducibility is correlated with lung cancer.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

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Aryl hydrocarbon hydroxylase (AHH) was present in explant cultures of human prostate obtained from surgery of benign prostatic hyperplasia and was inducible by benz[a]anthracene (BA). The induction of AHH ranged from 14- to 150-fold when compared with control values and 10-fold variation of AHH inducibility among individuals was observed. Epithelial cells grown from human prostate tissue also contained measurable AHH activity and AHH was inducible by BA and 7,12-dimethylbenz[a]anthracene (DMBA). Inducibility of AHH by BA ranged from 2- to 63-fold. The inducibility of AHH by DMBA was always less than that by BA. In cells treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), there were no changes in AHH activity. These findings support the view that the human prostate is susceptible to environmental polycyclic hydrocarbon carcinogens and that environmental and occupational factors might contribute to the etiology of human prostatic carcinoma.     

Isolation,X-Ray Structure and Biological Activity of Deacetyl-dihydrobotrydial Produced by Botrytis squamosa

Mitogenic substances on human peripheral blood mononuclear leukocytes were screened from culture filtrates of microorganisms newly isolated from soil and sea water by measuring [³H]- thymidine incorporation into the cells. Strong mitogenic activity was found in marine bacteria, particularly in marine vibrios. These mitogen samples exhibited neither hemagglutinating activity nor leukoagglutinating activity. They could scarcely stimulate murine lymphocytes.

Cell-cell interaction among leukocyte subsets in response to a bacterial mitogen was investigated using the most powerfully mitogenic sample (culture filtrate of strain H 52–2). A slight decrease in the mitogen response was observed on depletion of plastic surface adherent cells. Separation of T and non-T cells from each other by erythrocyte-rosette sedimentation resulted in a markedly diminished mitogen response. Considerable restoration of the mitogen response was obtained when T cells were mixed with mitomycin C-treated adherent cells or mitomycin C-treated non-T lymphocytes, or when non-T lymphocytes were mixed with mitomycin C-treated T cells.     

The immunological capacity of peripheral lymphocytes in a blast-transformation system using frozen-stored cells

Suspensions of isolated peripheral lymphocytes were frozen to ?95 °C using a programmed freezing apparatus and dimethyl sulphoxide as a cryoprotective agent. In comparisons among fresh cells, frozen cells, and cells stored in storage medium, freezing was found to be the best method of storage with retention of almost the same immunocapacity as fresh cells and with the same coefficients of variation for results after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, purified protein derivative, and allogenic cells in mixed lymphocyte cultures as was obtained with fresh cells. Blast transformation was found to be dependent on the number of cells in the cultures, the amount of [¹⁴C]thymidine added, and the amount of phytohemagglutinin used but independent of the amounts of pokeweed mitogen and concanavalin A. The maximum responses for normal lymphocytes and lymphocytes from uremic patients after stimulation with phytohemagglutinin occurred simultaneously, but after stimulation with allogenic cells maximum response was obtained earlier for “uremic” than for normal lymphocytes.It was concluded that frozen-stored lymphocytes are suitable for in vitro quantitative measurements of the cellular immune response in an immunological sequential study provided that the above mentioned factors are well defined.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Properties and activities of aminopeptidases in normal and mitogen-stimulated human lymphocytes.

Human peripheral lymphocytes were found to contain at least two distinct aminopeptidases, designated cytosol aminopeptidase and microsomal aminopeptidase, which differed from one another with respect to intracellular localization, substrate specificity, metal-ion activation, Km value and electrophoretic mobility. No change in these aminopeptidase activities was observed in cultured lymphocytes in the absence of mitogen throughout the cultivation period. The addition of phytohaemagglutinin or concanavalin A to the culture medium caused, in dose-dependent manner, a significant increase in cytosol aminopeptidase activity in lymphocytes. On the other hand, no increase in microsomal aminopeptidase activity was observed under the same conditions. The biochemical properties of aminopeptidases in stimulated cultured lymphocytes were identical with those of the enzymes in peripheral lymphocytes and unstimulated cultured lymphocyte. The phytohaemagglutinin dose-response curves for lymphocyte activation as measured by the DNA synthesis rate and for cytosol aminopeptidase activity were observed to be similar. However, when DNA synthesis was temporarily blocked by hydroxyurea, the rate of increase of aminopeptidase activity was unaffected. Pokeweed mitogen only slightly increased the cytosol aminopeptidase activity in cultured lymphocytes, although the lymphocytes were highly activated.     

Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive indicator of exposure to ionizing radiation. However, this assay cannot be immediately applied after radiation exposure because expression of a mutant phenotype may require as long as several months. In the present study, we eliminate this time lag by stimulating lymphocytes with a mitogen that can accelerate the turnover of TCR protein expression in T-cells. When lymphocytes obtained from healthy donors were irradiated with various doses of X-rays and cultured with human interleukin-2 after phytohemagglutinin (PHA) pulse stimulation, the mutant frequency (MF) of CD4⁺ T-cells increased dose dependently during the first 7 days, then decreased rapidly due to the growth disadvantage of mutant cells. This suggests that PHA stimulation can shorten the expression time of a mutant phenotype to within a week after radiation exposure. The relationship between radiation dose and TCR MF on the seventh day was best fitted by a linear-quadratic dose–response model. We applied this improved TCR mutation assay to gynecological cancer patients who received 5 days of localized radiotherapy, totaling about 10 Gy. The in vivo TCR MF in the patients did not change within a week after radiotherapy, whereas the in vitro TCR MF of PHA-stimulated lymphocytes from the same patients significantly increased 7 days after initiating culture. The estimated mean radiation dose to the peripheral blood lymphocytes of the cancer patients was about 0.9 Gy, based on the in vitro linear-quadratic dose–response curve. This estimated dose was close to that described in a previous report on unstable-type chromosome aberrations from cervical cancer patients after receiving the same course of radiotherapy. On the basis of these findings, we propose that the improved TCR mutation assay is a useful biological dosimeter for recent radiation exposure.     

Human bone marrow lymphocytes. III. Polyclonal activation of B lymphocytes in human bone marrow measured by a direct plaque-forming cell assay.

Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.     

Effects of recombinant human interferon alpha on aryl hydrocarbon hydroxylase activity in cultured human peripheral lymphocytes

Interferon and its inducers are known to depress drug biotransformation in vivo by decreasing the levels of cytochrome P-450 (P450) monooxygenase system in the liver. However, very little is known about the effects of interferon on P450 in extrahepatic tissues. In this study we investigated the effects of a recombinant human interferon-alpha (rhIFN-alpha) on aryl hydrocarbon hydroxylase (P450IAI) in cultured human peripheral lymphocytes (HPL). Non-induced and induced (3-methylcholanthrene) mitogen activated lymphocytes were used throughout the study. rhIFN-alpha maximally depressed AHH activity to approximately 58% of control after 24 hrs of incubation in both non-induced and induced lymphocytes. However, after 48 hrs of incubation with rhIFN-alpha, AHH activity had recovered to 86% of control in induced cells and 61% in non-induced cells. rhIFN-alpha had no significant effect on either NADH cytochrome c reductase activity or on viable lymphocyte cell count. This is the first demonstration that rhIFN-alpha can have a direct depressive effect on a P450 dependent monooxygenase system in HPL.     

Genetics of aryl hydrocarbon hydroxylase induction in mice: Response of the lung to cigarette smoke and 3-methylcholanthrene

When mice from different inbred strains are injected intraperitoneally with 3-methylcholanthrene (MC), the activity of aryl hydrocarbon hydroxylase (AHH) rapidly increases in livers of some strains but not others. AHH plays a role in the metabolism of polycyclic hydrocarbons. Alleles at a small number of loci account for most of the variation in inducibility of hepatic AHH among mice, when MC is used as the inducing agent. Cigarette smoke is a common source of carcinogenic polycyclic hydrocarbons in the environment. Since some of the hydrocarbons in cigarette smoke are metabolized by AHH, the activity of AHH in tissues may affect the carcinogenicity of smoke in those tissues. The purpose of these experiments was to see whether induction of AHH in lung in response to cigarette smoke is regulated by the same genes that regulate induction of AHH in liver in response to MC. Mouse strains AKR/J and C57L/J and six recombinant inbred (RI) lines derived from them were tested for the response of AHH in lung and liver to parenteral MC or inhalation of cigarette smoke. Inducibility (the ratio of MC-induced AHH activities to basal AHH activities) in liver from MC-treated RI lines is bimodal and compatible with Mendelian segregation of genes at a small number of loci. Increased activities of AHH in MC-treated liver are associated with increased ability to metabolize BP and whole smoke condensates to mutagens detected by Salmonella typhimurium TA1538. Inducibility of AHH in lung in response to MC is not bimodal, and no definite conclusion about the number of loci can be made. When actual levels of AHH activity are considered, following the administration of MC as inducing agent, there is a correlation (r=0.89, p<0.01) between AHH levels in liver and lung, suggesting that some genes affecting liver also affect lung. Basal and MC-induced AHH levels in lung are also correlated (r=0.86, p<0.01). Mice with high basal activities have two to threefold higher levels of AHH after MC treatment than do mice with low basal activities. Induction of AHH in pulmonary tissues occurs in all mice after either parenteral MC or smoke inhalation. In contrast to MC treatment, AHH activities in lungs following smoke inhalation are not correlated with AHH levels in liver after MC (r=0.49) and are only weakly correlated with basal (r=0.66, 0.05相似文献   

Low molecular weight factors displaying augmenting activity for human antibody production in vitro

Dialyzable low molecular weight antibody-augmenting factors (LMAAF) were found in the culture supernatant of human tonsillar lymphocytes which were not stimulated by antigen and/or mitogen in vitro. Phagocyte-depleted nylon wool-adherent lymphocytes (M-Ny+ cells) were responsible for the release of the LMAAF. Marbrook's culture system was adopted to assay for the LMAAF. The M-Ny+ cells, which were cultured without antigen and/or without mitogen in the reservoir of Marbrook's diffusion culture vessel, released the LMAAF, which diffused across a dialysis membrane and significantly augmented the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) response of phagocyte-depleted lymphocytes (M-cells) cultured in the inner vessel. Phagocyte-depleted nylon wool-passed lymphocytes (M-Ny- cells) cultured in the reservoir could not augment the PWM-induced PFC response of the M- cells cultured in the inner vessel. The exuded fluid, which was the dialysate of the culture supernatant of the M-Ny+ cells ultrafiltrated with dialysis tubing, also enhanced the PFC response of M- cells cultured in 24-well multi plates. The exuded fluid also augmented the total IgM and IgG production of human tonsillar and peripheral blood lymphocytes measured by enzyme-linked immunosorbent assay (ELISA) systems. Gel filtration chromatography on Sephadex G-25 Superfine column showed that the LMAAF activity was demonstrated in the fractions corresponding to a molecular weight (m.w.) of 362 to 1,355 and a m.w. of 3,560 to 5,700, with a peak activity at about 4,500 dalton. The LMAAF were inactivated by treatment with proteinase K, but not by trypsin, alpha-chymotrypsin, RNase, and DNase, and were stable when treated at 56 C for 60 min. The dialysates of culture supernatants from two out of seven Epstein-Barr virus (EBV)-transformed M-Ny+ cell lines showed LMAAF-like activity. These results indicate that phagocyte-depleted nylon wool-adherent lymphocytes, possibly B cells, release low molecular weight factors displaying augmenting activity for human antibody production in vitro.     

Generalised tissue abnormality of aryl hydrocarbon hydroxylase in psoriasis.

Microsomal aryl hydrocarbon hydroxylase (AHH) activity and inducibility were measured in jejunal mucosa, liver, and lesion-free epidermis of patients with psoriasis. In all three tissues AHH activity and inducibility were less than in controls. This demonstration of a generalised enzymatic abnormality in the tissues of patients with psoriasis is in keeping with the suggestion that it may be close to the underlying genetic defect.     

Suppressor cell influence in selected strains of inbred rats: I. Strain-dependent mitogen- and antigen-related low responsiveness

Lymphocytes derived from Lewis (LE), Brown-Norway (BN), or the F₁ hybrid (LBNF₁) rats respond in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. The magnitude of the response, as determined by incorporation of ³H-thymidine, ¹⁴C-adenine, and ³H-leucine, was highest for LE and lowest for BN animals. These proliferation response differences were observed for lymph node lymphocytes and peripheral blood lymphocytes. The response to antigen, as measured by lymphocyte transformation, reflected the mitogen responsiveness of the strains tested, i.e., LE animals responded to a higher level than did BN animals. Equivalent levels of antibody were found in all animals immunized with antigen. In addition, BN rats are suppressed to a greater magnitude than are LE rats when both strains are primed, rechallenged, and assayed via lymphocyte transformation to the test antigen.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.