Messenger RNA for glutamine synthetase: its partial purification, translation in a cell-free system and its regulation by hydrocortisone.

We have verified that the enzymatic hydroxylation of carcinogenic aromatic hydrocarbons produces product molecules in electronically excited states. Introduction of the carcinogen benzo[α]pyrene into liver microsomes from 3-methylcholanthrene-induced rats results in a significant chemiluminescence which is shown for the first time to be correlated with the enzymatic hydroxylation of the parent carcinogen. The kinetics of the chemiluminescence implicate the intermediate epoxide as the precursor to the excited state product molecules.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T12; CL8 cell line

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T$\text{1}\text{2}$ CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent K_m-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 μM BP, 3.3 nM NADPH) than in confluent cells (0.74–1.39 μM BP, 33.4–53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the K_m-value for BP was less in log phase (0.2–0.4 μM BP) than in confluent cells (0.64–1.05 μM BP). Moreover, the K_m-values for BP and NADPH in control cultures at confluency (0.10–0.14 μM BP, 15.4–23.2 nM NADPH) were less than those for BA-treated cells (0.64 μM BP, 37.9–54.8 nM NADPH) under the same nutritional conditions. The finding that the K_m-value for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.     

Protection against 3-methylcholanthrene-induced skin tumorigenesis in Balb/C mice by ellagic acid

Topical application of ellagic acid, a naturally occurring dietary plant phenol, to Balb/C mice resulted in significant protection against 3-methylcholanthrene (MCA)-induced skin tumorigenesis. Ellagic acid was found to be an effective inhibitor of tumor formation whether the tumor data are considered as percent mice with tumors, cumulative number of tumors, tumors per mouse or tumors per tumor bearing animal as a function of the number of weeks on test. By 8, 10, 12, 14, and 16 weeks of testing, the number of tumors per mouse in the group receiving MCA alone was 2.0, 3.4, 4.0, 4.9 and 5.3, respectively, whereas the corresponding numbers in the group receiving MCA plus 2 mumol ellagic acid were 0, 0.3, 0.4, 0.6 and 1.2, respectively. At the termination of the experiment (16 weeks) aryl hydrocarbon hydroxylase (AHH) activity in skin and liver and the extent of 3H-BP-binding to skin, liver and lung DNA were determined and both of these parameters were found to be significantly inhibited in the animals treated with ellagic acid. These results indicate that ellagic acid can inhibit the metabolism of polyaromatic hydrocarbons and modulate skin carcinogenesis induced by these chemicals.     

CYP1A1 gene polymorphisms and smoking status as modifier factors for lung cancer risk

Objective

Lung cancer remains the most prevalent malignancy worldwide. Susceptibility to lung cancer has been shown to be modulated by inheritance of polymorphic genes. Several metabolic enzymes are currently under investigation for their possible role in lung cancer susceptibility, including members of the cytochrome P450 (CYP) superfamily. The aim of this work was to identify the correlation between CYP1A1 m1 and m2 polymorphisms and lung cancer risk and figure its interactions with smoking as genetic modifiers in the etiology of lung cancer in the Egyptian population.

Materials and methods

One hundred and ten patients with lung cancer and one hundred and ten controls were enrolled in the study. CYP1A1 m1 and m2 polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism.

Results

Subjects carrying TC and CC genotypes of CYP1A1 m1 and AG and GG genotypes of CYP1A1 m2 were significantly more likely to develop lung cancer especially squamous cell carcinoma. The proportion of lung cancer attributable to the interaction of smoking and CYP1A1 m1 and CYP1A1 m2 polymorphisms was 32% and 52% respectively.

Conclusion

Our results revealed that CYP1A1 m1 and m2 polymorphisms contribute to smoking related lung cancer risk in the Egyptian population.     

Metabolic activation of promutagens, detectable in Ames' Salmonella assay, by 5000 X g supernatant of rat ventral prostate

Induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (7-EOD) activities as well as of benzo[a]pyrene (BP) metabolite formation in rat prostatic microsomes has been demonstrated after treatment with beta-naphthoflavone (BNF). The capacity to convert promutagenic compounds to ultimate mutagenic metabolites in the Ames' Salmonella assay by 5000 X g supernatant of rat ventral prostate was investigated. Male rats were treated with BNF, polychlorinated biphenyls (PCB; Arochlor 1254), phenobarbital (PB) and the vehicle, corn oil. PCB or BNF pretreatment increased the AHH- and 7-EOD activities 100-200-fold in the rat prostate 5000 X g supernatant (S-5 fraction). Epoxide hydrolase (EH) and glutathione-S-transferase (GST) activities were not affected while UDP-glucuronosyltransferase (UDP-GT) was increased 2.2- and 2.5-fold by PCB and BNF, respectively. PB did not significantly affect any of the enzyme activities measured. A dose-dependent increase in mutagenic response versus amount of 5000 X g supernatant and promutagen (aflatoxin B1 (AFB), 2-aminofluorene (2-AF), BP) was observed. The most pronounced activation was obtained with S-5 fraction from BNF- or PCB-treated rats. The great sensitivity of prostatic AHH to certain inducers and the capacity of the prostate to produce mutagenic metabolites might be of importance for initiation of prostatic cancer by environmental factors.     

Regulation of the binding of 7,12-dimethylbenz[a]-anthracene to DNA of cultured murine epidermal cells at metabolic level: competition of the binding by polycyclic aromatic hydrocarbons and by other precarcinogens.

The binding of labeled carcinogen [3H]DMBA to murine epidermal cells (MEC) DNA in culture has been studied. The influence of unlabeled noncarcinogenic and carcinogenic polycyclic aromatic hydrocarbons (PAH), several PAH metablites, and various directly and indirectly acting non-PAH carcinogens on the binding of [3H]DMBA to MEC DNA has been examined. All the carcinogenic PAH and some of non-carcinogenic PAH effectively inhibit the binding of [3H]DMBA to MEC DNA. The non-PAH chemical carcinogens requiring metabolic activation also reduce the binding of labeled DMBA to MEC DNA; however, a higher concentration of these compounds is required for 50% inhibition of binding than the concentrations of PAH for the same degree of inhibition of binding of [3H]DMBA to MEC DNA. The directly acting carcinogens do not significantly inhibit the binding of [3H]DMBA to DNA. The relationship between structures of PAH and their ability to inhibit the binding of [3H]DMBA to MEC DNA is also discussed. Thus, it appears that the binding of DMBA to cellular DNA is primarily controlled at a level of metabolism and to some extent at the level of binding of reactive metabolites to DNA.     

Enhancement of DNA binding,mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[a]pyrene (BP) metabolism and activation and, to a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).     

Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit

The in vivo formation of benzo[alpha]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.     

Factors for efficiency of the Salmonella/microsome mutagenicity assay.

Factors were studied which modify the enzymatic capacity of mouse liver microsomal mixed-function oxidase to convert vinylidene chloride (1.1-dichloroethylene) (VDC) into mutagens in the Salmonella/microsome mutagenicity test. A microsomal fraction incorporated in soft agar layer converted VDC into mutagens during 7 h at a constant rate; these were detected with S. typhimurium TA100. In absence of VDC the enzymatic activity declined gradually to nil after 14 h of incubation at 37 degrees C. The presence of EDTA greatly enhanced the microsome-mediated mutagenicity of VDC and led to prolonged enzymatic viability, but only when liver fractions from phenobarbitone (PB) pretreated mice were used. The efficiency of the plate incorporation assay for the detection of mutagens is discussed in comparison with assays in liquid suspension.     

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.