Is the comet assay a sensitive procedure for detecting genotoxicity?

Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were exactly identical; (3) the power of the Comet assay to detect a low level of genotoxic potential can be elevated to a level higher than that of MN test by using DNA resynthesis inhibitors, such as araC and HU.

A candidate cDNA clone for (-)-limonene-7-hydroxylase from Perilla frutescens.

Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (-)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (-)-limonene. The perilla cDNA was a partial clone of 1550bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively.

Production of the monoterpene limonene and modulation of apoptosis-related proteins in embryonic-mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia).

The monoterpene D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia). Apoptosis was not observed in the limonene-producing cells. A concomitant increase in the level of apoptosis-related protein Bcl-2 (B-cell lymphoma protein 2) and decreases in the levels of Bad (Bcl-2 antagonist of cell death) and phosphorylated JNK (c-Jun N-terminal kinase) were observed in limonene-producing cells. Limonene-producing cells may provide a useful new system to investigate the in vivo function of this monoterpene.

Anticarcinogenic compounds in the Uzbek medicinal plant, Helichrysum maracandicum.

An ethanol extract of Helichrysum maracandicum showed antiproliferative activity against cultured cells of SENCAR mouse in an in vitro assay, and activity-guided fractionation of the extract resulted in the isolation of isosalipurposide as an active substance. Naringenin chalcone, the aglycone of isosalipurposide, also showed strong antiproliferative activity. An in vivo assay of two-stage carcinogenesis on mouse skin revealed that epidermal application of isosalipurposide resulted in delayed formation of papillomas. Western blot analysis showed that the expression of p38 mitogen-activated protein kinase was suppressed by the administration of naringenin chalcone or isosalipurposide, which might be related to the anticarcinogenic activity.

Perilla frutescens var. frutescens in northern Laos.

Twenty-eight samples of mericarps of Perilla frutescens var. frutescens were collected through fieldwork performed in Phongsali and Xieng Khouang provinces in northern Laos. No perilla samples were collected from Savannakhet province in the south although more than 20 sites were investigated. Perilla plants are mostly grown mixed with dry-paddy rice by slash-and-burn cultivation in Laos. The most popular local name for perilla mericarps in the area was "Ma Nga Chan". Weight of 1,000 grains and hardness of the mericarps were measured, and all mericarps were found to be large (weight of 1,000 grains around 2 g) and soft (limit load weight under 300 g), which were preferred for culinary use in Laos. The composition of the essential oils obtained from the herbaceous plants raised from the mericarps was divided into five types, perillaketone, elemicine plus myristicine, shisofuran, piperitenon, and myristicine, and GC-MS analysis of these Laotian perilla samples showed that they were similar to those of corresponding types of known Japanese perilla strains. One of the shisofuran-type perilla contained large amounts of putative alpha-naginatene, which is likely to be an intermediate of the biosynthesis of naginataketone. The farmers' indifference to the oil type of the leaf seems to leave Laotian perilla as a good genetic resource for studies of the biosynthesis of oil compounds.

Sedative effects of vapor inhalation of agarwood oil and spikenard extract and identification of their active components.

Agarwood oil and spikenard extract were examined for their sedative activity using a spontaneous vapor administration system. It was shown that inhalation of agarwood oil vapor sedated mice. The main volatile constituents of the oil were found to be benzylacetone [agarwood oil from a Hong Kong market (1)], or alpha-gurjunene and (+)-calarene [agarwood oil made in Vietnam (2)]. A hexane extract of spikenard contained a lot of calarene, and its vapor inhalation had a sedative effect on mice. Individual principles benzylacetone, calarene, and alpha-gurjunene were administered to mice, which reproduced the result of the corresponding oil or extract. However, the most effective dose of the compounds was lower than their original content in the oil and extract (benzylacetone 0.1%, calarene 0.17%, alpha-gurjunene 1.5%).

A new phenolic glucoside from an Uzbek medicinal plant, Origanum tyttanthum.

A new phenolic glucoside was isolated from the aerial parts of Origanum tyttanthum, an Uzbek medicinal plant, together with 12 known compounds. The structure of the new compound was elucidated as 4-O-beta-D-glucopyranosylbenzyl-3'-hydroxyl-4'-methoxybenzoate (1) based on the spectral and chemical evidence.

Sesquiterpene lactones from the roots of Ferula varia and their cytotoxic activity.

The ethyl acetate-soluble fraction from a MeOH extract of the roots of Ferula varia gave six new sesquiterpene lactones (1-6) and five known sesquiterpenes (7-11). Their structures were established on the basis of spectroscopic evidence. The cytotoxic activities of 1-11 were evaluated against selected human cancer cell lines. Compound 4 showed significant selective cytotoxicity against multidrug-resistant cancer cells (KB-C2). The cytotoxicities of compounds 1, 3, 5, 8, and 11 against KB-C2 cells were enhanced in the presence of nontoxic concentrations of colchicine.

Geraniol synthases from perilla and their taxonomical significance.

Geraniol synthases were isolated from five pure strains of Perilla citriodora and Perilla frutescens which vary in essential oil type, the main compounds of which were citral, elsholtziaketone, perillaketone, and perillene, respectively. This result supports the putative biosynthetic pathways of these three furylalkenes which are all produced by way of citral. Nucleotide sequences of geraniol synthases from three oil types of P. citriodora were identical, and almost the same as the sequence from P. frutescens, a species with twice the chromosome number of P. citriodora. This identity in sequence between P. citriodora and P. frutescens, together with other previous results, indicates that P. frutescens was formed as an amphidiploid of P. citriodora and an unknown wild species.

Antichagasic activity of komaroviquinone is due to generation of reactive oxygen species catalyzed by Trypanosoma cruzi old yellow enzyme.

A novel potent trypanocidal diterpene, komaroviquinone, was reduced by Trypanosoma cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. Since TcOYE is expressed throughout the T. cruzi life cycle, komaroviquinone is an interesting candidate for developing new antichagasic drugs.

Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton.

A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.

Suppressive mechanisms of Sairei-to on mesangial matrix expansion in rat mesangioproliferative glomerulonephritis.

BACKGROUND: Sairei-to (TJ-114) is a Japanese herbal medicine of standardized quality, originating from traditional Chinese medicine. In the present in vivo study, we investigated the suppressive effects of TJ-114 and related drugs, Shosaiko-to (TJ-9), and Saiboku-to (TJ-96), on mesangioproliferative glomerulonephritis (MsPGN) in rats. TJ-9 is a basal prescription of TJ-96 and TJ-114. We evaluated the efficacy of these drugs on proteinuria, extracellular matrix (ECM) accumulation, and superoxide dismutase (SOD)-activity. METHODS: MsPGN in Wistar rats was induced by intravenous injection of rabbit anti-rat thymocyte serum (ATS). TJ-114, TJ-9, TJ-96 (500 mg/kg/day), or prednisolone (PSL, 2 mg/kg/day) was orally administered to the rats as drinking water from the day of ATS injection (day 0) to day 8, when rats were sacrificed and the kidney specimens were collected. Macrophage infiltration was evaluated by immunostaining for ED-1. ECM was measured by trichrome-staining, and fibronectin immunostaining. Northern blotting was performed to clarify the mRNA expression of cytokines and fibronectin. SOD-activity in the homogenate of renal cortex was also evaluated. RESULTS: The amount of urinary protein was significantly decreased only in the TJ-114-treated group compared with the disease control group (p < 0.05). The number of ED-1-positive cells was significantly decreased in all the treatment groups (p < 0.05, respectively). Decreases in the trichrome-stained area were observed moderately in the TJ-114-treated group (66% of control, p < 0.001) and mildly in the PSL-treated group (76% of control, p < 0.001). The staining area of fibronectin in the glomerulus was significantly decreased in all the treated groups except PSL, and was especially suppressed in the TJ-114-treated group (45% of control, p < 0.001). Transforming growth factor (TGF) and connective tissue growth factor (CTGF) expression significantly decreased in the TJ-114-treated group to the control level (p < 0.05). TGF-beta, CTGF, and fibronectin mRNA were upregulated in the disease control group, and TJ-114 suppressed these mRNA expressions in glomeruli. The SOD-activity of renal cortex-homogenate was significantly augmented in all the treated groups except PSL, markedly in the TJ-96- and TJ-114-treated groups. CONCLUSION: These results suggest that TJ-114 ameliorates ECM accumulation in experimental rat MsPGN, partly suppressing TGF-beta and CTGF expression through the recovery of SOD-activity.

Bioactive constituents from Dracocephalum subcapitatum (O. Kuntze) Lipsky.

From an EtOAc extract of Dracocephalum subcapitatum, five flavonoids, calycopterin, xanthomicrol, isokaempferide, luteolin and apigenin, together with five terpenoids, oleanolic acid, ursolic acid, geranial, neral and limonene-10-al, were isolated. Among them, citral and limonene-10-al were the most effective components against epimastigotes of Trypanosoma cruzi, the parasitic agent of Chagas disease.

cDNA isolation and functional expression of myrcene synthase from Perilla frutescens.

cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase. The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P. frutescens (85% identity). Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene. As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.

New norditerpenoids with trypanocidal activity from Vitex trifolia.

Trypanocidal constituents of the fruits of Vitex trifolia were investigated. Activity-guided isolation of the acetone extract resulted in the isolation of two new norditerpene aldehydes, 1 and 2, together with five known diterpenes: vitexifolin E (3), vitexifolin F (4), vitexilactone (5), 6-acetoxy-9-hydroxy-13(14)-labden-16,15-olide (6), and previtexilactone (7). In vitro minimum lethal concentrations of the isolated compounds against epimastigotes of Trypanosoma cruzi were 11 microM (1), 36 microM (2), 34 microM (3), 34 microM (4), 66 microM (5), 66 microM (6), and >265 microM (7).

New sesquiterpene hydroperoxides with trypanocidal activity from Pogostemon cablin.

Trypanocidal constituents of Pogostemon cablin were investigated. Activity guided isolation of the acetone extract resulted in isolation of three new sesquiterpene hydroperoxides 1-3, together with a known sesquiterpene, patchouli alcohol (4). In vitro minimum lethal concentrations of the hydroperoxides 1-3 against epimastigotes of Trypanosoma cruzi were 0.84 microM (1), 1.7 microM (2) and 1.7 microM (3). The activity of the corresponding alcohols and patchouli alcohol was very weak (MLC>200 microM).

Prenylated benzophenones and xanthones from Hypericum scabrum.

Two new polyprenylated benzophenones, a new polyprenylated phloroglucinol, and six new xanthone derivatives were isolated from the aerial parts of the Uzbekistan medicinal plant Hypericum scabrum. Their structures were elucidated on the basis of spectroscopic evidence. The isolated compounds showed moderate cytotoxicity for human tumor cells.

Two new monoterpene glycosides and trypanocidal terpenoids from Dracocephalum kotschyi.

From the whole plant of Dracocephalum kotschyi BOISS., two new monoterpene glycosides (9, 10), together with seven known terpenoids and a phytosterol (1-8), were isolated. Their structures were determined to be limonen-10-al (1), geranial (2), neral (3), beta-sitosterol (4), oleanolic acid (5), ursolic acid (6), p-mentha-8-en-1,2-diol (7), colosolic acid (8), limonen-10-ol 10-O-beta-D-glucopyranoside (9), and limonen-10-ol 10-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside (10). Compounds 1 (3.1 microM), 2 (3.1 microM), 3 (3.1 microM), 5 (6.2 microM), 6 (6.2 microM), and 8 (6.2 microM) were effective against epimastigotes of Trypanosoma cruzi.

Suppressive effects of Sairei-to on mesangial proliferation in a rat model of glomerulonephritis.

BACKGROUND: Sairei-to (TJ-114) is a Japanese herbal medicine of standardized quality, originating from traditional Chinese medicine. In the present in vivo study, we investigated the suppressive effects of TJ-114, Shosaiko-to (TJ-9), and Saiboku-to (TJ-96) on mesangioproliferative glomerulonephritis (MsPGN) in rats. We evaluated the efficacy of these drugs on proteinuria, mesangial cell proliferation, and superoxide dismutase (SOD) activity. METHODS: MsPGN was induced in Wistar rats by intravenous injection of rabbit anti-rat thymocyte serum (ATS). TJ-114, TJ-9, or TJ-96 (500 mg/kg per day) was orally administered to the rats in drinking water from the day of ATS injection (day 0) to day 8, when rats were killed and kidney specimens were collected. The degree of mesangial cell proliferation was evaluated by immunostaining for proliferating cell nuclear antigen (PCNA) or macrophage antigen (ED-1). SOD activity in the homogenate of the renal cortex was also evaluated. RESULTS: The amount of urinary albumin was significantly decreased only in the TJ-114-treated group compared with the disease control group ( P < 0.05). The number of PCNA- or ED-1-positive cells was significantly decreased in all the treatment groups ( P < 0.05, respectively, compared with the disease control group). SOD activity in the renal cortex homogenate was significantly augmented in all the treatment groups, most markedly in the TJ-96- and TJ-114-treated groups ( P < 0.01, respectively, compared with the disease control group). CONCLUSIONS: These results suggest that TJ-114 suppresses the proliferation of mesangial cells through its antioxidative activity.

Role of mesangial Factor V expression in crescent formation in rat experimental mesangioproliferative glomerulonephritis.

It has been suggested that fibrin deposition participates in the development of crescents in active glomerulonephritis (GN). In human IgA nephropathy, which is a common form of mesangioproliferative GN (MsPGN), crescent formation is occasionally observed in active disease, leading to end-stage renal failure. Factor V is a membrane-bound potent cofactor for the conversion of prothrombin to thrombin by Factor Xa. An in vivo study was conducted to clarify the contribution of local fibrin production to crescent formation in MsPGN through mesangial Factor V expression. Wistar rats were injected intravenously with rabbit anti-rat thymocyte serum. Three days after injection, mesangiolysis with intense mesangial Factor V expression was observed and immunoelectron microscopy revealed fibrin localization in mesangiolytic lesions, which had spread into the glomerular basement membrane adjacent to the destroyed mesangium, accompanied by clots in Bowman's space. Marked glomerular fibrin deposition, together with its deposition in Bowman's space and cellular crescent formation, was noted with mesangial proliferation on day 8. Specific bands for Factor V mRNA were also detected from isolated glomeruli. Fibrin deposition and cellular crescent formation were significantly suppressed by treatment with anti-Factor V antibody. These results suggest that local fibrin production, following mesangial Factor V expression, together with mesangiolysis that spreads to the adjacent glomerular basement membrane, plays a role in crescent formation in MsPGN.