Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain

The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.     

Cytochrome P450 1 genes in early deuterostomes (tunicates and sea urchins) and vertebrates (chicken and frog): origin and diversification of the CYP1 gene family

Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.     

Effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro

Colon cancer is the third most frequent cancer in humans. Carica papaya leaves are vegetable foods consumed by most people around the world; it has potential as an anticancer. Therefore it is of interest to investigate the effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro. Human Colon cancer cell line (HT-29) was purchased from the National Centre for Cell Sciences, Pune, India. Cell viability test was done by MTT assay. Gene expression analysis was done by Real Time-PCR. The obtained data were analyzed statistically by one-way analysis of variance and Duncan''s multiple range test with Graph Pad Prism version 5 to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan''s test. Carica papaya caused a marked increase in cell death in a dose dependent manner. At the end of 48 hours, maximum inhibition was at 300 and 400 µg/ml. Carica papaya has significantly reduced the mRNA expression of Wnt and beta catenin (p<0.05). Data showed that Carica papaya leaf extract has anticancer activity on Colon cancer cell lines (HT-29).     

Fasciola hepatica: Morphological changes in vitelline cells following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD)

Fasciola hepatica: morphological changes in vitelline cells following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD). International Journal for Parasitology 18: 1061–1069. The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml⁻¹) on the vitelline cells of Fasciola hepatica over an 18 h period in vitro has been determined by transmission electron microscopy. DAMD acts preferentially on the undifferentiated stem cells and the intermediate cells in the early stages of protein synthesis; it appears to prevent their continued development. In the stem cell the nucleolus is absent after 6 h. During the rest of the drug treatment period considerable clumping of heterochromatin takes place, the cells round up and become electron-dense. Signs of autophagy are also evident, and the mitochondria become swollen and disorganized. From 6 h onwards there are progressive changes in the It1 (intermediate type 1) cells, including clumping of the heterochromatin in the nucleus, a decrease in the number of egg-shell globules (and consequently a reduction in the number and size of the shell globule clusters), and a decrease in the number of ribosomes on the GER cisternae, although the GER system remains well-developed. By 18 h the nucleolus is absent, and the cells are very rounded and electron-dense; the mitochondria are swollen and disorganized. Similar changes are evident in the It2 (intermediate type 2) cells, so that by 18 h it is difficult to distinguish between the It1 and It2 cells. In the mature cells there is a progressive decrease in the number and size of the shell globule clusters from 9 h onwards. Glycogen synthesis and ‘yolk’ formation may also be impaired and lipid droplets are present. Spaces begin to appear between the nurse cell cytoplasm and the vitelline cells after 9 h, and disruption of the nurse cell cytoplasm is evident after 12 h, becoming very severe by 18 h. By this time the follicle is very disorganized and empty-looking. In more severely affected follicles the mature cells are seen to be breaking down. Over the 18 h drug treatment period, a change in the cell population of the follicle takes place, with relatively more stem, early It1 and mature cells being present, whilst few if any characteristic It1 and It2 cells remain. The results are interpreted as being due to an inhibition of protein synthesis in the vitelline cells by DAMD.     

Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with β-naphthoflavone-treated fish

Induction of cytochrome P4501A (CYP1A) in fish is an important biomarker in marine monitoring programmes but a number of factors complicate interpretation of data based on catalytic activity. To provide additional analytical tools, we have cloned and sequenced entire (dab) and partial cDNAs (flounder, turbot, sand eel) from several fish species. A detailed analysis comparing the new sequences to those on the database (13 sequences) is presented and identifies an invariant, teleost-specific sequence (195-IVVSVANVICGMCFGRRYDH-214) which might be the basis for production of a species cross-reactive antibody. Northern and slot blots of fish RNA (sand eel, plaice, turbot, flounder and dab) showed extensive cross-species hybridisation with each of the cDNAs (sand eel, plaice, turbot, flounder and dab). The exception was turbot RNA, which only gave adequate hybridisation when the turbot probe was used. Attempts to normalise the hybridisation data to GAPDH mRNA were not satisfactory since there were significant species differences in expression of this gene and expression was suppressed (20–40%) by β-naphthoflavone treatment. The CYP1A probes indicated induction levels relative to untreated dab of: plaice (five-fold); turbot (12-fold); flounder (12-fold); and dab (10-fold). The study demonstrates the relative ease with which species-specific molecular probes can be generated and used.     

Microspectrofluorimetric study of the kinetics of cellular uptake and metabolization of benzo(a)pyrene in human T 47 D mammary tumor cells: evidence for cytochrome P1450 induction

The kinetics of penetration, activation and detoxification of benzo(a)pyrene were determined by near U.V. microspectrofluorimetric measurements on single living cells. This technique allows one to monitor the different intracellular fluorescent species present in a subcellular microvolume by using spectral decomposition of the fluorescence data. The T47-D cell line was chosen for its high capability of metabolization. The penetration involves a simple diffusion transfer through the cytoplasmic membrane of the cell, with a half-time of 2 min. The metabolization process gives rise, with more than a one hour delay after intracellular incorporation of the hydrocarbon, to a rapid conversion of B(a)P into unconjugated metabolites, leading to a transient accumulation of the 3OH-B(a)P metabolite in the cell. This feature may be related to the enhancement of cytochrome P₁450 activity, induced by the B(a)P itself. The ability of the cell to increase its Cyt-P₁450 level, after exposure to B(a)P, gives indirect evidence for the presence of the Ah gene complex in the T47-D cell line.Abbreviations B(a)P benzo(a)pyrene - PAH polycyclic aromatic hydrocarbon - AHH aryl hydrocarbon hydroxylase - (+)-antiB(a)PdE (+)-7 - 8-dihydroxy-9 10-epoxy 7,8,9,10-tetrahydrobenzo(a)pyrene - MSF microspectrofluorimetry - DHD dihydrodiol Offprint requests to: F. Sureau     

BimEL-dependent apoptosis induced in peripheral blood lymphocytes with n-butyric acid is moderated by variation in expression of c-myc and p21(WAF1)

We have examined the effect of sodium butyrate (SB) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and the effect of this agent on the expression of 20 apoptosis-related genes. Data suggest that PBL treated with 2 mmol L(-1) SB resisted for at least 8 h the destructive activity of the agent, but eventually 30% of cells died within 72 h. As documented by flow cytometry and cytochrome c release study, cells underwent mitochondrial-derived apoptosis. While the expression of the majority of genes examined by RT-PCR and Western blots remained indifferent to 2 mmol L(-1) SB, the cellular levels of BimEL, c-myc, p53, and p21(WAF1) varied profoundly with the time of SB treatment. The Bax activator BimEL increased rapidly, driving cells toward apoptosis likely controlled by c-myc and p21(WAF1) activities. The c-myc, exercising the role of mediator of the function of BimEL and inhibitor of p21(WAF1) expression, decreased significantly for several hours after adding SB but within 48 h it returned to close to its original value. An apoptosis inhibitor and executive caspase substrate p21(WAF1) increased early at the beginning of treatment but subsequently, within a time frame of 72 h, profoundly dropped in terms of both a caspase-dependent and caspase-independent way. We suggest that variations in c-myc and p21(WAF1) expression delay apoptosis making PBL resistant to SB for several hours, and together with fast catabolism of SB in vivo protect PBL against the destructive activity of this anti-cancerous metabolite of colonic bacteria.     

Comparative excretion and tissue distribution of selenium in mice and rats following treatment with the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate

In a previous preliminary investigation, we reported on the excretion, tissue disposition and metabolism of the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) in the rat, but similar studies in the mouse have not been explored. Following the oral administration of p-XSC (50 micromol/kg body weight), selenium excretion in feces was comparable to that in urine in mice, but in rats, feces was the major route of excretion. Tetraselenocyclophane (TSC) was the major metabolite detected in mouse and rat feces. In both species, levels of selenium in exhaled air were negligible. At termination, in the mouse, the stomach had the highest selenium content followed by liver and blood, but lung and kidney contained negligible levels of selenium; in the rat, the selenium level in liver was the highest followed by kidney, stomach, blood and lung. The identification of TSC as a fecal metabolite in both species let us to postulate the following metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->a selenol (p-XSeH)-->TSC. Since the glutathione conjugate appears to be the proximal precursor for the selenol metabolite that may be an important intermediate in cancer chemoprevention, we report for the first time the synthesis of p-XSeSG and its other potential metabolites, namely the cysteine- and N-acetylcysteine-conjugates of p-XSC. HPLC analysis of the urine and bile showed a few metabolites of p-XSC; none of which eluted with the synthetic standards described above. When we examined the conversion of p-XSC and p-XSeSG in vitro using rat cecal microflora, TSC was formed from p-XSeSG but not from p-XSC. The formation of TSC from p-XSC in vivo but not in vitro suggests that p-XSC needs to be metabolized to p-XSeSG or an intermediate derived from its further metabolism. Thus, p-XSeSG was given orally to rats and the results showed that the pattern of selenium excretion after p-XSeSG treatment was similar to that of p-XSC; TSC was also identified as a fecal metabolite of p-XSeSG. It may be that the conversion of p-XSeSG to TSC is too facile, or the mere conjugation of p-XSC with glutathione does not occur in rats and mice.     

Recombinant human (rh) stem cell factor and rhIL-4 stimulate differentiation and proliferation of CD3+ cells from umbilical cord blood and CD3+ cells enhance FcepsilonR1 expression on fetal liver-derived mast cells in the presence of rhIL-4

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcepsilonR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 +/- 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4(-)CD8-. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcepsilonR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells.     

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.     

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.     

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C⁶⁰⁹T variant genotypes was observed among MS patients compared to controls (P = 0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P = 0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P = 0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C⁶⁰⁹T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.