PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Bioactive C17 and C18 Acetylenic Oxylipins from Terrestrial Plants as Potential Lead Compounds for Anticancer Drug Development.

Bioactive C17 and C18 acetylenic oxylipins have shown to contribute to the cytotoxic, anti-inflammatory, and potential anticancer properties of terrestrial plants. These acetylenic oxylipins are widely distributed in plants belonging to the families Apiaceae, Araliaceae, and Asteraceae, and have shown to induce cell cycle arrest and/or apoptosis of cancer cells in vitro and to exert a chemopreventive effect on cancer development in vivo. The triple bond functionality of these oxylipins transform them into highly alkylating compounds being reactive to proteins and other biomolecules. This enables them to induce the formation of anti-inflammatory and cytoprotective phase 2 enzymes via activation of the Keap1-Nrf2 signaling pathway, inhibition of proinflammatory peptides and proteins, and/or induction of endoplasmic reticulum stress, which, to some extent, may explain their chemopreventive effects. In addition, these acetylenic oxylipins have shown to act as ligands for the nuclear receptor PPARγ, which play a central role in growth, differentiation, and apoptosis of cancer cells. Bioactive C17 and C18 acetylenic oxylipins appeartherefore, to constitute a group of promising lead compounds for the development of anticancer drugs. In this review, the cytotoxic, anti-inflammatory and anticancer effects of C17 and C18 acetylenic oxylipins from terrestrial plants are presented and their possible mechanisms of action and structural requirements for optimal cytotoxicity are discussed.

Mitomycin C induces fibroblast apoptosis and reduces intra-articular scar adhesion by regulating miR-21 and its target Programmed cell death 4.

Previous studies have shown that mitomycin C (MMC) can prevent scar adhesion after joint surgery, but the specific mechanism underlying this effect remains unclear. The purpose of this study was to explore the specific mechanism by which MMC promotes fibroblast apoptosis and prevents joint adhesion. The effect of MMC on fibroblasts was assessed using cell counting kit-8 (CCK-8) assays, western blotting, and TUNEL staining. We used qRT-PCR to measure the expression of miR-21 in fibroblasts treated with MMC. Luciferase activity assays were used to determine the relationships between miR-21 and Programmed cell death 4 (PDCD4). The effects of miR-21 and PDCD4 on fibroblast apoptosis were assessed using flow cytometry and western blotting. HE staining was used to determine the role of miR-21 in scar tissue formation in a model of joint adhesion. The results showed that MMC induced apoptosis of fibroblasts and decreased the expression of miR-21. Moreover, miR-21 down-regulation also induced apoptosis of fibroblasts. PDCD4 was confirmed to be a direct target of miR-21 by luciferase activity assay. The results from the animal model indicated that miR-21 attenuated the effect of MMC on reducing the number of fibroblasts. Our study shows that MMC can induce fibroblast apoptosis and prevent joint adhesion by regulating the expression of miR-21 and its target PDCD4.

In vivo induction of hepatocellular carcinoma by diethylnitrosoamine and pharmacological intervention in Balb C mice using Bergenia ciliata extracts / Indução in vivo de carcinoma hepatocelular por dietilnitrosoamina e intervenção farmacológica em camundongos balb c usando extratos de Bergenia ciliata

Abstract Background Hepatocellular carcinoma is the most frequent primary malignancy of liver and accounts for as many as one million deaths worldwide in a year. Objectives The aim of the present study was to evaluate the anti-cancerous efficiency of Bergenia ciliata rhizome against diethylnitrosoamine induced hepatocarcinogenesis in Balb C mice. Methods One percent diethylnitrosoamine was prepared by using 99 ml of normal saline NaCl (0.9 percent) solution to which was added 1 ml of concentrated diethylnitrosoamine (DEN) solution (0.01 μg/μl). Extract of Bergenia ciliata was prepared by maceration technique. Mice were classified into four groups as follows: Group 1 a control group (N=7) received saline solution (3.5 μl/mg), group 2 (N=14) received diethylnitrosoamine (3.5 μl/mg) intraperitoneally once in a week for eight consecutive weeks, group 3 (N=7) received plant extract (150 mg/kg (Body weight)) once in a week, while group 4 (N=7) was given combination of diethylnitrosoamine (3.5 μl/mg) and plant extract (150 mg/kg (Body weight)). After eight weeks of DEN induction group 2 mice were divided into two subgroups containing seven mice each, subgroup 1 was sacrificed while subgroup 2 was treated with plant extract (150 mg/kg (Body weight)) once in a week for eight consecutive weeks. Results The model of DEN injected hepatocellular carcinomic (HCC) mice elicited significant decline in levels of albumin with concomitant significant elevations in tumor markers aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alpha feto protein (AFP), gamma glutamyl transferase (Y-GT), 5 nucleotidase (5NT), glucose-6-phosphate dehydrogenase (G6PDH) and bilirubin. The intraperitoneal administration of B. ciliata as a protective agent, produced significant increase in albumin levels with significant decrease in the levels of tumor markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alpha feto protein (AFP), gamma glutamyl transferase (Y-GT), 5 nucleotidase (5NT), glucose-6-phosphate dehydrogenase (G6PDH) and bilirubin. Conclusion Bergenia ciliata has potent antioxidant activity, radical scavenging capacity and anticancerous properties. Bergenia ciliata extracts may provide a basis for development of anti-cancerous drug.

Resumo Antecedentes O carcinoma hepatocelular é a neoplasia primária mais frequente do fígado e é responsável por até um milhão de mortes em todo o mundo em um ano. Objetivos O objetivo do presente estudo foi avaliar a eficiência anticancerígena do rizoma de Bergenia ciliata contra a hepatocarcinogênese induzida por dietilnitrosoamina em camundongos balb c. Métodos Um por cento de dietilnitrosoamina foi preparado usando 99 ml de solução salina normal (0,9 por cento) à qual foi adicionado 1 ml de solução concentrada de dietilnitrosoamina (DEN) (0,01 μg / μl). O extrato de Bergenia ciliata foi preparado pela técnica de maceração. Os ratos foram classificados em quatro grupos: Grupo 1 grupo controle (N = 7) recebeu solução salina (3,5 mL / mg), grupo 2 (N = 14) recebeu dietilnitrosoamina (3,5 mL / mg) por via intraperitoneal uma vez por semana para oito semanas consecutivas, o grupo 3 (N = 7) recebeu extrato vegetal (150 mg / kg (peso corporal)) uma vez por semana, enquanto o grupo 4 (N = 7) recebeu combinação de dietilnitrosoamina (3,5 μl / mg) e extrato (150 mg / kg (peso corporal). Após oito semanas do grupo de indução DEN 2 ratos foram divididos em dois subgrupos contendo sete ratos cada, subgrupo 1 foi sacrificado enquanto subgrupo 2 foi tratado com extrato vegetal (150 mg / kg)) uma vez por semana durante oito semanas consecutivas. Resultados O modelo de camundongos hepatocelulares carcinômicos (CHC) injetados com DEN provocou declínio significativo nos níveis de albumina com elevações significativas concomitantes nos marcadores tumorais: aspartato aminotransferase, alanina aminotransferase (ALT), lactato desidrogenase (LDH), proteína alfa feto (AFP), gama glutamiltransferase (Y-GT), 5 nucleotidase (5NT), glicose-6-fosfato ehidrogenase (G6PDH) e bilirrubina. A administração intraperitoneal de B. ciliata como agente protetor produziu um aumento significativo nos níveis de albumina com uma diminuição significativa nos níveis dos marcadores tumorais: aspartato aminotransferase, alanina aminotransferase (ALT), lactato desidrogenase (LDH), proteína alfa feto (AFP), gama glutamiltransferase (Y-GT), 5 nucleotidase (5NT), glicose-6-fosfato desidrogenase (G6PDH) e bilirrubina. Conclusão Bergenia ciliata possui atividade antioxidante potente, capacidade de eliminação de radicais livres e propriedades anticancerígenas. Extratos de Bergenia ciliata podem fornecer uma base para o desenvolvimento de drogas anti-cancerígenas.

In vivo induction of hepatocellular carcinoma by diethylnitrosoamine and pharmacological intervention in Balb C mice using Bergenia ciliata extracts.

BACKGROUND: Hepatocellular carcinoma is the most frequent primary malignancy of liver and accounts for as many as one million deaths worldwide in a year. OBJECTIVES: The aim of the present study was to evaluate the anti-cancerous efficiency of Bergenia ciliata rhizome against diethylnitrosoamine induced hepatocarcinogenesis in Balb C mice. METHODS: One percent diethylnitrosoamine was prepared by using 99 ml of normal saline NaCl (0.9 percent) solution to which was added 1 ml of concentrated diethylnitrosoamine (DEN) solution (0.01 µg/µl). Extract of Bergenia ciliata was prepared by maceration technique. Mice were classified into four groups as follows: Group 1 a control group (N=7) received saline solution (3.5 µl/mg), group 2 (N=14) received diethylnitrosoamine (3.5 µl/mg) intraperitoneally once in a week for eight consecutive weeks, group 3 (N=7) received plant extract (150 mg/kg (Body weight)) once in a week, while group 4 (N=7) was given combination of diethylnitrosoamine (3.5 µl/mg) and plant extract (150 mg/kg (Body weight)). After eight weeks of DEN induction group 2 mice were divided into two subgroups containing seven mice each, subgroup 1 was sacrificed while subgroup 2 was treated with plant extract (150 mg/kg (Body weight)) once in a week for eight consecutive weeks. RESULTS: The model of DEN injected hepatocellular carcinomic (HCC) mice elicited significant decline in levels of albumin with concomitant significant elevations in tumor markers aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alpha feto protein (AFP), gamma glutamyl transferase (Y-GT), 5 nucleotidase (5NT), glucose-6-phosphate dehydrogenase (G6PDH) and bilirubin. The intraperitoneal administration of B. ciliata as a protective agent, produced significant increase in albumin levels with significant decrease in the levels of tumor markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alpha feto protein (AFP), gamma glutamyl transferase (Y-GT), 5 nucleotidase (5NT), glucose-6-phosphate dehydrogenase (G6PDH) and bilirubin. CONCLUSION: Bergenia ciliata has potent antioxidant activity, radical scavenging capacity and anticancerous properties. Bergenia ciliata extracts may provide a basis for development of anti-cancerous drug.

Hibiscus acetosella extract protects against alkylating agent-induced DNA damage in mice.

Hibiscus acetosella was shown to exert beneficial effects in humans and animal models however, the effects of this plant on DNA are unknown. The aim of this study was to determine the antigenotoxic and antimutagenic effects of H. acetosella extracts on alkylating agent methyl methanesulfonate (MMS) in vivo in mice. Initially, we performed analysis of phenolic compounds in extracts of H. acetosella by high-performance liquid chromatography (HPLC). Next, mice were divided into 8 groups and treated with distilled water or plant extract (0.1 ml/10 g) by gavage for 15 days, followed by intraperitoneal (ip) administration of saline solution or MMS (40 mg/Kg b.w) on day 16. Caffeic acid, following by gallic acid, gallocatechin, coumaric acid, and 3,4-dihydroxybenzoic acid were found to be present in extracts of H. acetosella leaves. In peripheral blood analysis of groups receiving pretreatment with H. acetosella at doses of 50 or 100 mg/kg plus MMS decreased DNA damage as evidenced by comet assay and Micronucleus assays relative to MMS alone. These results suggested that H. acetosella extracts exerted protective effects dose dependent against genotoxicity and mutagenicity induced by alkylating agents.

Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin-Antibody Conjugate.

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab-IR700 (pan-IR700) and the NIR-releasing compound, CyEt-panitumumab-duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor-background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661-70. ©2017 AACR.

DNA damage response in neonatal and adult stromal cells compared with induced pluripotent stem cells.

UNLABELLED: Comprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest. SIGNIFICANCE: The results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent stem cells (iPSCs). The data suggest developmental age-dependent differences in DNA damage repair capacity. In iPSCs (closest to embryonic stem cells), the highest expression level of DNA damage response and repair genes was found, followed by neonatal stromal cells and adult stromal cells with the lowest overall expression. In addition, a differentiation-dependent downregulation of repair capacity was observed during osteogenic differentiation in neonatal stromal cells. Notably, the impact of genotoxic stress on osteogenic differentiation depended on the time the genotoxic insult took place and, moreover, was agent-specific. These results strongly support the necessity of offering and establishing adequate cell sources for informative toxicological testing matching to the developmental age and differentiation status of the respective cell of interest.

A forward genetic screen with a thalamocortical axon reporter mouse yields novel neurodevelopment mutants and a distinct emx2 mutant phenotype.

BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.

Astrocytic poly(ADP-ribose) polymerase-1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity.

Genotoxicity study of Hypiran and Chamomilla herbal drugs determined by in vivo supervital micronucleus assay with mouse peripheral reticulocytes.

The growth in popularity of Over The Counter (OTC) of medicinal products or other natural sources have taken a very large share of healthcare system therefore it is essential to determine their safety as for as public health is concerned.In this study Maximum Tolerated Dose (MTD) was obtained according to CSGMT protocol presented by the Environmental Mutagen Society of Japan. The positive group received mitomycin C in dose of 0.5 mg/kg. The peripheral blood samples before treatment (zero time) were considered as negative control. The MTD of Hypiron was 12 ml/kg and for Chamomilla was 10 ml/kg. Doses of MTD, 1/2 MTD and 1/4 MTD were considered for test groups. Then blood samples were prepared 48 hours after first administration of drugs and kept on precoated Acridine orange slides. The scoring of micronucleated reticulocytes were carried out per 2000 counted reticulocytes in each slide by fluorescent microscope. The results were statistically analyzed. Results of Hypiran were compared with negative control group and the P value was P > 0.05, but the p value of Chamomilla was P < 0.05. Also, the p value of Hypiran and Chamomilla compared with historical negative control group was less, therefore Chamomilla herbal drog can be classified as equivocul category of genotoxicity and Hypiran did not show genotoxicity.

Mixed tocopherols prevent mammary tumorigenesis by inhibiting estrogen action and activating PPAR-gamma.

PURPOSE: Tocopherols are lipophilic antioxidants present in vegetable oils. Although the antioxidant and anticancer activities of alpha-tocopherol (vitamin E) have been studied for decades, recent intervention studies with alpha-tocopherol have been negative for protection from cancer in humans. The tocopherols consist of four isoforms, which are the alpha, beta, gamma, and delta variants, and recent attention is being given to other isoforms. In the present study, we investigated the inhibitory effect of a tocopherol mixture rich in gamma- and delta-tocopherols against mammary tumorigenesis. EXPERIMENTAL DESIGN: Female Sprague Dawley rats were treated with N-methyl-N-nitrosourea (NMU), and then fed diets containing 0.1%, 0.3%, or 0.5% mixed tocopherols rich in gamma- and delta-tocopherols for 9 weeks. Tumor burden and multiplicity were determined, and the levels of markers of inflammation, proliferation, and apoptosis were evaluated in the serum and in mammary tumors. The regulation of nuclear receptor signaling by tocopherols was studied in mammary tumors and in breast cancer cells. RESULTS: Dietary administration of 0.1%, 0.3%, or 0.5% mixed tocopherols suppressed mammary tumor growth by 38%, 50%, or 80%, respectively. Tumor multiplicity was also significantly reduced in all three mixed tocopherol groups. Mixed tocopherols increased the expression of p21, p27, caspase-3, and peroxisome proliferator activated receptor-gamma, and inhibited AKT and estrogen signaling in mammary tumors. Our mechanistic study found that gamma- and delta-tocopherols, but not alpha-tocopherol, activated peroxisome proliferator activated receptor-gamma and antagonized estrogen action in breast cancer. CONCLUSION: The results suggest that gamma- and delta-tocopherols may be effective agents for the prevention of breast cancer.

Cutaneous biotransformation of N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane and its product, 4-bromobenzamide, leading to percutaneous penetration enhancement of drugs: initial evidence using hydrocortisone.

The role of the skin's metabolism of N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane (DMBIS), an effective penetration enhancer, on its enhancement activity was investigated. It has been found that DMBIS hydrolyzes very fast in physiological buffer to 4-bromobenzamide (BBA), and even faster and almost completely in the presence of skin tissue. It was further shown that in the presence of skin from different species incubated at physiological conditions, the concentration of BBA (DMBIS' immediate product) dropped sharply to 70-80% in 10 min followed by a slower decrease of 0.35-0.50 microg/h. This metabolism was partially inhibited by a continuous application of iodine, and more profoundly, by iodoacetic acid (IAA) and dithiothreitol (DTT) combination treatment. This indicates that at least a part of the metabolism of BBA involves enzymes that are sensitive to reactions with their sulfhydryl groups. In an in vitro permeation study using human epidermis and conventional diffusion cells, we compared between the permeabilities of untreated epidermis and IAA/DTT-treated epidermis to hydrocortisone in the presence of BBA. Due to its metabolic inhibition, we noted a higher penetration of BBA through IAA/DTT-treated epidermis than through the untreated epidermis. Contrary to these results, the extent of the penetration of hydrocortisone was higher through the untreated epidermis with only 1.6 h lag time relative to its penetration through IAA/DTT-treated epidermis, which exhibited a lag time of 12.4 h. It is evident, therefore, that the skin enhancement activity of DMBIS/BBA depends on BBA metabolism in the skin, presumably through its in situ biotransformation into an active enhancer.

Borrelia burgdorferi vlsE antigenic variation is not mediated by RecA.

RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

DNA sequence selective adenine alkylation, mechanism of adduct repair, and in vivo antitumor activity of the novel achiral seco-amino-cyclopropylbenz[e]indolone analogue of duocarmycin AS-I-145.

AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins.

Protective effects of guarana (Paullinia cupana Mart. var. Sorbilis) against DEN-induced DNA damage on mouse liver.

Guarana (Paullinia cupana Mart. var. Sorbilis) is a plant originally from Brazil, which is rich in tannins. Some tannins are known to present protective effects against DNA damage. This study was performed to investigate the anti-genotoxic/cytotoxic properties of guarana in hepatocytes of mice injected with N-nitrosodiethylamine (DEN). The protective effect of guarana was evaluated both by comet assay and DNA smear fragmentation technique in two month-old female BALB/c mice. These were treated previously with 2.0 mg/g bw of guarana for 16 days and then injected with DEN (160 microg/g body weight) to induce DNA damage. The DEN-only treated group presented higher comet image length than the guarana plus DEN and untreated groups (116.06+/-5.0 microm, 104.09+/-3.3 microm and 93.28+/-14.4 microm, respectively; p<0.01). Guarana treatment presented a 52.54% reduction in comet image length when animals were exposed to DEN (p<0.05). DNA samples from the guarana plus DEN group clearly showed less EtBr fluorescence intensity when compared to the DEN-only group, reinforcing the comet assay data. These results show, for the first time, that guarana has a protective effect against DEN-induced DNA damage in mouse liver.

The effects of thyme volatiles on the induction of DNA damage by the heterocyclic amine IQ and mitomycin C.

The leafy parts of thyme and its essential oil have been used in foods for its flavour, aroma and preservation for many years. In the present study the genotoxic potential of major compounds of thyme oil, i.e. thymol, carvacrol, and gamma-terpinene and of the methanolic extracts of thyme, were investigated in human lymphocytes by single-cell gel electrophoresis. Also, the effects of these substances on the induction of DNA damage by 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and mitomycin C (MMC) were evaluated. No increase in DNA strand breakage was observed at thymol and gamma-terpinene concentrations below 0.1 mM, but at the higher concentration of 0.2 mM significant increases in DNA damage were seen. Thymol and gamma-terpinene significantly reduced the DNA strand breakage induced by IQ and MMC at the lower concentrations studied. Carvacrol, which is an isomer of thymol, seemed to protect lymphocytes from the genotoxic effects of IQ and MMC at non-toxic concentrations below 0.05 mM, but at the higher concentration of 0.1 mM carvacrol itself induced DNA damage. Also the constituents of the n-hexane and ethyl acetate fractions prepared from the concentrated aqueous methanolic extracts of Thymus spicata protected lymphocytes against IQ- and MMC-induced DNA damage in a concentration-dependent manner.

Suppression of diethylnitrosamine and 2-acetylaminofluorene-induced hepatocarcinogenesis in rats by tocotrienol-rich fraction isolated from rice bran oil.

The anticancer efficacy of tocotrienol-rich fraction (TRF) was evaluated during diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)-induced hepatocarcinogenesis in male Sprague-Dawley rats. TRF treatment was carried out for 6 months, and was started 2 weeks before initiation phase of hepatocarcinogenesis. Morphological examination of the livers from DEN/AAF rats showed numerous off-white patches and few small nodules, which were significantly reduced by TRF treatment. Cytotoxic damage by DEN/AAF was estimated by alkaline phosphatase (ALP) release into the plasma from the cell membranes. DEN/AAF caused a twofold increase in the activity of ALP in plasma as compared with normal control rats, and this increase was prevented significantly by TRF treatment. We observed an increase of 79% in liver ALP activity in DEN/AAF rats, which was further increased by another 48% after the administration of TRF. Hepatic activity of glutathione S-transferase (GST) was also increased (3.5-fold) during the induction of hepatic carcinogenesis. Lipid peroxidation and low-density lipoprotein (LDL) oxidation increased threefold following initiation by DEN/AAF as compared with normal control rats. However, TRF treatment to DEN/AAF-treated rats substantially decreased (62-66%) the above parameters and thus limited the action of DEN/AAF. We conclude that long-term intake of TRF could reduce cancer risk by preventing hepatic lipid peroxidation and protein oxidation damage due to its antioxidant actions.

Clusterin as a biomarker in murine and human intestinal neoplasia.

Early detection of colorectal cancer is critical for the management of this disease. Biomarkers for early detection of several cancers have been developed and applied clinically in recent years. We have sought to discover candidate biomarkers without the restricted choice of markers placed on microarrays, and without the biological complications of genetic and environmental heterogeneity. We have compared by cDNA subtraction two genetically matched sets of mice, one developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then subjected to a series of validation steps. In situ hybridization and immunohistochemistry were used to analyze clusterin expression at a cellular level on a series of murine intestinal and human colonic neoplasms. Elevated clusterin expression was characterized within certain regions of murine and human tumors regardless of tumor stage, location, or mode of initiation. The cells showing high clusterin levels generally lacked differentiation markers and adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed low levels of clusterin. Its specific expression patterns and correlation with cellular events during tumorigenesis make it a useful diagnostic tool in the mouse and a potential contributor to the set of biomarkers for early detection of human colon cancer.

Sodium selenite, dietary micronutrient, prevents the lymphocyte DNA damage induced by N-nitrosodiethylamine and phenobarbital promoted experimental hepatocarcinogenesis.

Selenium (Se), a micronutrient, has a long history in chemoprevention of mammary and colon cancers in rodent models. Se is a current clinical trial, having shown promise in prevention of prostate and other human cancers. The mechanisms involved in the in vivo anti-carcinogenic activity of Se remain to be elucidated. In the present study, we examined the effect of sodium selenite supplementation in lymphocytes, obtained from hepatoma bearing rats on DNA damage in correlation with oxidative stress. In addition, this study examined the supplementation of Se at 4-ppm levels in the form of sodium selenite either before initiation or during initiation and/or promotion phase's increases lymphocyte Se concentrations. This in turn improves lymphocyte resistance to oxidative stress and protection against the lymphocytes DNA damage. Supplementation of Se increased lymphocyte Se concentration and reduced lymphocytes DNA damage as determined by single cell gel electrophoresis. The enzymatic antioxidants such as superoxide dismutase, glutathione peroxidase, and catalase were found to be decreased while the thiobarbituric acid reactive substances level was increased in the lymphocytes of hepatoma bearing rats. Furthermore, the reactive oxygen species such as superoxide radicals and hydroxyl radicals were also found to be high in lymphocytes. Our present results explain the understanding of unique association between anti-peroxidative effect of Se and ultimately the capability of Se to prevent cancer.