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Bioactive substances of plant origin in food--impact on genomics.

In the past decade, substantial progress has been made concerning our knowledge of bioactive components in plant foods and their links to health. Human diets of plant origin contain many hundreds of compounds which cannot be considered as nutrients, but appear to play a role in the maintenance of health. These substances are called nutraceuticals. In some cases where the disease process is at least partially understood, elements of protection can be related to a single compound or structurally related group of compounds in the diet. Bioactive components of food which are of special interest include the following groups: polyphenols, phytoestrogens, phytosterols, phytates and polyunsaturated fatty acids. Most of them are featured by antioxidant properties. In the first part of this review, we indicate the main groups of bioactive compounds giving a description of their localisation, chemical properties and biological actions. Recently, it was shown, however, that the bioavailability of potential antioxidants from plant foods is generally too low to have any substantial direct effect on reactive oxygen species. As a result of that it is postulated that dietary compounds, even in very low concentrations, may have a far greater impact than previously appreciated on the regulation of gene expression. The second part of this paper concerns the action of the literally most important bioactive substances on the molecular mechanisms of the control of genes which in turn affect cellular metabolism. A few current studies on the action of selected nutraceuticals on the activity of transcription factors such as AP-1, NF-kappaB, SREBPs, PPARs as final targets in the signal transduction cascade and gene regulation are included. A detailed analysis of numerous factors of dietary origin with their targets is far beyond the scope of this paper. However, continuing research on the effects of nutraceuticals on gene expression should provide insight into the mechanisms of prevention of diseases such as obesity, diabetes, atherosclerosis, hypertension and cancer by dietary manipulations.

Animals↗

Persistently low immunoreactive and normally bioactive plasma LH during male puberty.

Low basal and LRH-stimulated (50 mcg/m2 i.v.) levels of plasma immunoreactive (IR) LH were repeatedly found in 8 boys aged 13 to 16 years who had been referred because of delayed onset of puberty (basal IR-LH 0.19 +/- 0.1 and peak 0.48 +/- 0.2 vs. 0.24 +/- 0.09 and 1.71 +/- 0.9 mIU/ml in matched normal controls, respectively). Upon termination of puberty (no more than 3.5 years after referral) IR-LH levels were still low in all 8. Using a rat Leydig cell bioassay system with LER-907 (NPA) as standard, LH bioactivity in these patients was compared with that in 8 matched controls (basal LH was 0.32 +/- 0.33 and 0.11 +/- 0.07, and peak levels 0.8 +/- 0.47 and 1.22 +/- 0.44 mIU/ml respectively). The ratio of basal LH bioactivity to IR-LH was higher in the patients (1.24 +/- 0.95) than in controls (0.47 +/- 0.26, P less than .05) as was that of peak bioactivity (1.89 +/- 1.2 vs. 0.83 +/- 0.35, P less than .05). In the three patients tested during sleep IR-LH levels showed no significant change. Basal plasma testosterone levels were appropriate for pubertal stage (400 +/- 80 ng/dl) and in the four patients tested following prolonged LRH stimulation (500 mcg i.v. over 3 hours) increased to 530 +/- 60 ng/dl. It is concluded that in some boys there may be consistently low plasma levels of IR-LH in association with normal LH bioactivity.

Adolescent↗

Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity.

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the beta-bulge (QGEESN) of IL-1 beta at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the beta-bulge was co-expressed with a second substitution (His-54 --> Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Amino Acid Sequence↗

Proteolytic cleavage of chromogranin A (CgA) by plasmin. Selective liberation of a specific bioactive CgA fragment that regulates catecholamine release.

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.

Amino Acid Sequence↗

Effect of additives on encapsulation efficiency, stability and bioactivity of entrapped lysozyme from biodegradable polymer particles.

Low encapsulation efficiency, incomplete and erratic release profiles are the most common features of controlled released protein delivery systems employing biodegradable polymers. In the present study, lysozyme as a model protein was encapsulated in biodegradable microspheres using solvent evaporation method and the effect of amphiphilic stabilizer, a basic salt and a lyoprotectant on microparticle formulation was evaluated. Incorporation rat serum albumin (RSA) in the internal aqueous phase during emulsion increased the encapsulation efficiency of lysozyme and maintained the bioactivity. Use of NaHCO3 improved the encapsulation efficiency of lysozyme from 15-94%, but at the cost of reduced in vitro release characteristics. Incorporation of both RSA and NaHCO3 improved the bioactivity of lysozyme and decreased burst release of the protein from the polymer particle, but reduced the encapsulation efficiency from 90-70%. Addition of sucrose in the internal aqueous phase lowered the encapsulation efficiency which was restored by its addition in the external aqueous phase. Maintenance of internal aqueous phase pH close to the iso-electric point of the protein and osmotic balance between the internal aqueous phase and the external aqueous phase during solvent evaporation method helped in better encapsulation of the protein drug. In vitro release of the lysozyme correlated with the effect of different excipients on entrapment in polymer matrix. Entrapment efficiency as high as 76%, low burst effect and high bioactivity of the entrapped lysozyme was observed from the polymer particles. Use of RSA, sucrose and NaHCO3 helped in a co-operative way towards the formulation of particles entrapping bioactive lysozyme.

Absorption↗

Assessment of the bioactive conformation of the farnesyltransferase protein binding recognition motif by computational methods.

Ras farnesyltransferase catalyzes the carboxyl-terminal farnesylation of Ras as well as other proteins involved in signal transduction processes. Previous studies demonstrated that its inhibition suppresses the activity of Ras transformed phenotypes in cultured cells, causing tumor regression in animal models. This observation led to the consideration of farnesyltransferase as a target for cancer therapy. In the present work we report the results of a computational study aimed at assessing the bioactive conformation of the peptide Cys-Val-Phe-Met, known to be the minimum peptide sequence that inhibits farnesyltransferase. For this purpose the conformational preferences of four analogs of the peptide were assessed by means of thorough searches of their respective conformational spaces, using a simulated annealing protocol as sampling technique. Specifically, two active analogs: Cys-Val-Tic-Met and Cys-Val-psi(CH2NH)Tic-Met and two inactive analogs: Cys-Val-Tic-psi(CH2NH)Met and Cys-Val-Aic-Met were selected for the present study. Low energy conformations of the four analogs were classified according to their structural motifs. The putative bioactive conformation of the minimum farnesyltransferase recognition motif was assessed by cross-comparison of the different classes of conformations obtained for the two active and the two inactive analogs. The putative bioactive conformation is characterized by two structural motifs: i) a C14 pseudo-ring stabilized by a hydrogen bond between the amino group of Cys1 and the carboxylate group of Met4 and a C11 pseudo-ring involving the residues Cys1 and Tic3. In addition, the thiol group of Cys1 side chain of the bioactive conformation points to the carboxylate moiety of Met4.

Algorithms↗

Glutathione-dependent bioactivation of haloalkanes and haloalkenes.

Haloalkanes and haloalkenes constitute an important group of widely used chemicals that have the potential to induce toxicity and cancer. The toxicity of haloalkanes and haloalkenes may be associated with cytochromes P450- or glutathione transferase-dependent bioactivation. This review is concerned with the glutathione- and glutathione transferase-dependent bioactivation of dihalomethanes, 1,2-dihaloalkanes, and haloalkenes. Dihalomethanes, e.g., dichloromethane, and 1,2-dihaloethanes, e.g., 1,2-dichloroethane and 1,2-dibromoethane, undergo glutathione transferase-catalyzed bioactivation to give S-(halomethyl)glutathione or glutathione episulfonium ions, respectively, as reactive intermediates. Haloalkenes, e.g., trichloroethene, hexachlorobutadiene, chlorotrifluoroethene, and tetrafluoroethene, undergo cysteine conjugate beta-lyase-dependent bioactivation to thioacylating intermediates, including thioacyl halides, thioketenes, and 2,2,3-trihalothiiranes. With all of these compounds, the formation of reactive intermediates is associated with their observed toxicity.

Alkanes↗

Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants.

PURPOSE OF STUDY: To review the nature, extent, bioactivities, and clinical application of polysaccharides in Traditional Chinese Medicine (TCM) especially as adjuvants in cancer treatment. METHODOLOGY: Literature review. FINDINGS: Many fungal- and plant-derived bioactive polysaccharides with a broad range of immunomodulatory activities are found in TCM. Some such polysaccharides have been developed into drugs and show clinical efficacy in controlled trials while the majority of such compounds remain as nutraceuticals with only preliminary research. Such polysaccharides are generally nontoxic and possess other bioactivities such as inducing differentiation, stimulating hematopoiesis, antimetastasis, and antiangiogenesis, which make them ideal adjuvants in modern cancer therapy. CONCLUSION: Bioactive polysaccharides occur extensively in TCM herbs and are the basis of potentially useful application of TCM as an adjuvant in cancer therapies.

Adjuvants, Immunologic↗

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis.

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.

Alkaline Phosphatase↗

Bioavailability of flavan-3-ols and procyanidins: gastrointestinal tract influences and their relevance to bioactive forms in vivo.

There is considerable interest in the bioavailability of flavan-3-ols such as tea catechins and cocoa-derived procyanidin components of the diet and their bioactivity in vivo. Their hydrogen-donating abilities and their propensity for nitration make these compounds powerful scavengers of reactive oxygen and nitrogen species. In addition, recent evidence has suggested that these compounds may interact with redox-sensitive cell signaling pathways. However, their bioactivity in vivo will be dependent on the absorption and metabolism of these compounds after ingestion and the reducing properties of resulting metabolites. Many cell, animal, and human studies have shown that flavanol monomers, such as epicatechin, are extensively metabolised to O-methylated forms and/or conjugated to glucuronides and sulphates during absorption into the circulation. The cleavage of higher procyanidin oligomers to mixtures of monomer and dimer in the stomach may act to enhance the potential for their absorption in the small intestine as higher oligomers have very limited absorption. Studies suggest that the major bioactive forms of flavanol monomers and procyanidins in vivo are likely to be metabolites and/or conjugates of epicatechin. One such metabolite, 3'-O-methylepicatechin, has been shown to exert protective effects against oxidative stress-induced cell death. Future studies will continue to concentrate on the exact mechanism of action of the bioactive forms of flavan-3-ols in vivo.

Animals↗

Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with ionic dissolution products of 58S bioactive sol-gel glass.

Embryonic stem (ES) cells represent a potentially useful cell source for tissue regeneration. Previously, using factors known to enhance differentiation and mineralization of primary osteoblasts, we were able to generate cell populations enriched with osteoblasts from a murine ES cell source. Dexamethasone was a potent inducer of osteoblast differentiation and the timing of stimulation markedly increased the proportion of osteoblast lineage cells. This study examined whether inorganic stimuli derived from bioactive glasses could affect the differentiation of osteoblasts in an ES-cell based system. Previous work has demonstrated the ability of soluble ions released from bioactive glasses undergoing dissolution in vitro to stimulate gene expression characteristic of a mature phenotype in primary osteoblasts. We report here on the potential of soluble extracts prepared from 58S sol-gel bioactive glass to further enhance lineage-specific differentiation in murine ES cells. Differentiation of ES cells into osteogenic cells was characterized by the formation of multilayered, mineralized nodules. These nodules contained cells expressing the transcription factor runx2/cbfa-1, and deposition of osteocalcin in the extracellular matrix was detected by immunostaining. When differentiating cells were placed in an osteoblast maintenance medium supplemented with soluble extracts prepared from bioactive glass powders, we observed increased formation of mineralized nodules (98 +/- 6%, mean +/- SEM) and alkaline phosphatase activity (56 +/- 14%, mean +/- SEM) in a pattern characteristic of osteoblast differentiation. This effect of the glass extracts exhibited dose dependency, with alkaline phosphatase activity and nodule formation increasing with extract concentrations. Compared with medium supplemented with dexamethasone, which had previously been used to enhance osteoblast lineage derivation, the glass extracts were as effective at inducing formation of mineralized nodules by murine ES cells. When glass extracts were used in combination with dexamethasone, a further increase in the number of nodules was observed (110 +/- 16%; cf. 83 +/- 7% for dexamethasone alone). This study demonstrates the capacity of an entirely inorganic material to stimulate differentiation of ES cells toward a lineage with therapeutic potential in tissue-engineering applications.

Animals↗

Effect of high-fat intake on lactogenic hormone bioactivity in premenopausal women.

To determine if dietary fat affects the bioactivity of circulating prolactin and growth hormone (lactogenic hormones), a controlled feeding study was conducted in six premenopausal women over two complete menstrual cycles. After an initial 7-10 d dietary adjustment, subjects were randomly assigned to either low-fat (25% of calories) or high-fat (46%) diets. The diets were similar in proportions of energy, protein, P:S ratio, and cholesterol. All subjects completed both diets in a crossover experimental design. Fasting blood samples were collected on alternate days throughout the study. Plasma hormone bioactivity was assayed by the Nb2 lymphoma cell method. Luteal-phase lactogenic hormone bioactivity appeared to be higher (averaging 28%; p less than 0.07) in response to high- rather than low-fat diets. No differences were seen in levels of radioimmunoreactive prolactin or growth hormone. Substantial changes in short-term fat intake may alter the level of lactogenic hormone bioactivity in young women.

Adult↗

Modulation of human chorionic gonadotrophin bioactivity during the first trimester of pregnancy.

The objective of this study was to evaluate the bioactivity of human chorionic gonadotrophin (HCG) during first trimester pregnancy. This was done by means of a retrospective analysis of sera from patients with first trimester normal intrauterine and ectopic pregnancies. Serum samples were obtained from 38 women with amenorrhoea of <10 weeks. From these, 19 had a normal intrauterine pregnancy (IUP) and 19 an ectopic pregnancy (EP). Cases were allocated to either low serum immunoreactive HCG (HCGi), intermediate HCGi or high HCGi concentrations (HCGi <5000 mUI/ml, between 5000 and 40,000 mIU/ml and >40,000 mIU/ml respectively). HCGi and oestradiol were measured by enzyme immunoassays and bioactive HCG by the mouse Leydig cell bioassay. All results were analysed by analysis of variance and unpaired Student's t-test. There was a significant difference between bioactive to immunoreactive HCG ratios (b/i ratio) between the subgroups of low, intermediate and high HCGi concentrations. Lower b/i ratios were found when HCGi concentrations were high (HCG b/i mean +/- SEM: high subgroup, 0.33 +/- 0.07 versus low subgroup: 1.50 +/- 0.12; P < 0.0001). Furthermore, the b/i ratios were inversely correlated with oestradiol (P < 0.0001) and HCGi (P < 0.0001) concentrations but not with gestational age. There was no difference in the b/i ratios when comparing IUP with EP. It is concluded that, in first trimester pregnancies, there is a likely modulation of HCG bioactivity which is inversely correlated with HCGi and oestradiol concentration. The underlying mechanisms and their physiological relevance remain to be elucidated.

Animals↗

Relationship between follicle size and gonadotrophin surge attenuating factor (GnSAF) bioactivity during spontaneous cycles in women.

BACKGROUND: We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries. METHODS: To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined. RESULTS: Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations. CONCLUSIONS: The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.

Activins↗

Bioactive and inactive forms of tumor necrosis factor-alpha in spinal fluid from patients with meningitis.

Twenty-four patients, including 12 with meningitis, were admitted with meningeal symptoms and fever. Their serum and spinal fluid tumor necrosis factor-alpha (TNF alpha) levels were determined by ELISA. TNF alpha immunoreactivity was found in spinal fluid of 10 meningitis patients and one nonmeningitis patient, whereas 7 sera, 5 from meningitis patients, contained TNF alpha. Levels were significantly higher in spinal fluids than in serum samples, and the TNF alpha bioactivity of spinal fluids and sera was considerably below predictions based on ELISA measurements. Gel filtration chromatography demonstrated the presence of both polymeric (greater than 200 kDa) and oligomeric (10-40 kDa) TNF alpha in spinal fluid. Significant bioactivity was obtained only from samples containing oligomeric cytokine. In agreement with previous in vitro findings, these results strongly indicate that bioactive TNF alpha oligomers form inactive polymers and monomers, which could contribute to the observed in vivo discrepancies between immunoreactive and bioactive protein. Finally, the data support the concept of local central nervous system production of TNF alpha in meningitis.

Adolescent↗

Bioactivation of mitomycin C by xanthine dehydrogenase from EMT6 mouse mammary carcinoma tumors.

BACKGROUND: Mitomycin C is an antineoplastic antibiotic requiring bioactivation to an alkylating species or to an intermediate capable of generating oxygen radicals for its toxic effect. The enzymes responsible for the in vivo activation of mitomycin C have been proposed to include NADPH-cytochrome-c reductase, DT-diaphorase, and xanthine oxidase. PURPOSE: In this study, xanthine dehydrogenase, an enzyme structurally similar to xanthine oxidase, was assessed for its ability to activate mitomycin C. Partially purified xanthine dehydrogenase, from EMT6 mouse mammary tumors, was investigated for its ability to bioactivate mitomycin C under both aerobic and hypoxic conditions. METHODS: We conducted this analysis by measuring mitomycin C-induced oxygen consumption, alkylating potential, and mitomycin C consumption and metabolite formation as determined by high-pressure liquid chromatography analysis. RESULTS: Bioactivation of mitomycin C by xanthine dehydrogenase under both aerobic and hypoxic conditions gave rise to the formation of a metabolite, 2,7-diaminomitosene. Formation of this metabolite and alkylating ability were greater under hypoxic than under aerobic conditions and were increased when the pH was decreased from 7.4 to 6.0. Mitomycin C consumption was the same under both aerobic and hypoxic conditions and was independent of pH. Oxygen consumption studies showed that xanthine dehydrogenase-activated mitomycin C consumed oxygen at a much lower rate than xanthine oxidase-activated mitomycin C. CONCLUSIONS: Xanthine dehydrogenase-activated mitomycin C appears to be a good alkylating species but a relatively poor generator of reactive oxygen when compared with xanthine oxidase activation under aerobic conditions. IMPLICATION: Xanthine dehydrogenase may play an important role in the bioactivation of mitomycin C to an alkylating species under both aerobic and hypoxic conditions.

Animals↗

Recombinagenic activity of four compounds in the standard and high bioactivation crosses of Drosophila melanogaster in the wing spot test.

The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster was employed to determine the recombinagenic and mutagenic activity of four chemicals in an in vivo eukaryotic system. Two different crosses involving the wing cell markers mwh and flr(3) were used: the standard cross and a high bioactivation cross. The high bioactivation cross is characterized by a high constitutive level of cytochromes P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Three-day-old larvae derived from both crosses were treated chronically with the oxidizing agent potassium chromate and with the three procarcinogens cyclophosphamide, p-dimethylaminoazobenzene and 9,10-dimethylanthracene. From both crosses two types of progeny were obtained: marker-heterozygous and balancer-heterozygous. The wings of both genotypes were analysed for the occurrence of single and twin spots expressing the mwh and/or flr(3) mutant phenotypes. In the marker-heterozygous genotype the spots can be due either to mitotic recombination or to mutation. In contrast, in the balancer-heterozygous genotype only mutational events lead to spot formation, all recombination events being eliminated. The oxidizing agent potassium chromate was equally and highly genotoxic in both crosses. Surprisingly, the promutagen cyclophosphamide also showed equal genotoxicity in both crosses, whereas p-dimethylaminoazobenzene was negative in the standard cross, but clearly genotoxic in the high bioactivation cross. 9,10-Dimethylanthracene showed a rather weak genotoxicity in the high bioactivation cross. Analyses of the dose-response relationships for mwh clones recorded in the two wing genotypes demonstrated that all four compounds are recombinagenic. The fraction of all genotoxic events which are due to mitotic recombination ranged from 83% (9,10-dimethylanthracene) to 99% (p-dimethylaminoazobenzene). These results demonstrate that the wing spot test in Drosophila is most suited to the detection of recombinagenic activity of genotoxic chemicals.

Animals↗

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation.

Cytochrome b(5) (b(5)) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b(5)/CYP-competent mutagenicity tester bacteria to study the role of b(5) in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b(5), and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b(5)-1A2, BTC-b(5)-2A6 and BTC-b(5)-2E1, respectively. The relative content of b(5) with CYP and RED in these three BTC-b(5)-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b(5)-void strains to determine the effect of b(5) on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased ( approximately 1.5-fold) in the presence of b(5). The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased approximately 3- and 23-fold, respectively when the bacteria contained b(5). The CYP2A6-mediated mutagenicity of NNK increased approximately 9-fold when co-expressed with b(5). The stimulatory effect of b(5) on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b(5) with CYP2A6 or CYP2E1. This demonstrates the prominent role of b(5) in the bioactivation of this carcinogen.

Aryl Hydrocarbon Hydroxylases↗