Maria Grazia Evandri

Antimutagenic and mutagenic activities of some terpenes in the bacterial reverse mutation assay.

The mutagenic and antimutagenic effects of linalool, linalyl acetate and beta-caryophyllene were evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA 98 and TA 100, and on Escherichia coli WP2uvrA strains. Neither linalool nor beta-caryophyllene showed mutagenicity, but linalyl acetate induced a statistically significant increase in the number of revertant colonies in WP2uvrA, both with and without S9 mixture. Linalool was devoid of antimutagenic activity against 2-nitrofluorene (2NF), sodium azide (SA), methyl methane sulfonate (MMS) and 2-aminoanthracene (2AA). In contrast, beta-caryophyllene showed a strong antimutagenic activity against 2NF: at the maximum concentration tested (6.40mg/plate) the number of 2NF-induced revertant colonies was reduced by 83.9%. beta-Caryophyllene also showed to counteract the mutagenicity of SA (in TA 100), MMS and 2AA (in WP2uvrA): the effect was weak against SA (inhibition lower than 25%) and moderate against MMS and 2AA (up to 30.5%). The antimutagenic activity of beta-caryophyllene observed here suggests further studies to evaluate its possible chemopreventive properties.

Antimicrobial activity of Epilobium spp. extracts

The antimicrobial activity of the Epilobium angustifolium, E. hirsutum, E. palustre, E. tetragonum and E. rosmarinifolium ethanolic extracts was studied in vitro on Gram-positive and Gram-negative bacteria, yeasts and fungi. The cytotoxicity of the extracts was also evaluated using the Artemia salina test. All the extracts showed antimicrobial activity in a range of concentrations between 10 and 650 microgml of dry extract. E. angustifolium and E. rosmarinifolium had the most broad spectrum of action inhibiting bacteria, yeasts and fungi. The extracts were devoid of toxicity on Artemia salina within the range of antimicrobial concentrations, suggesting that the action is selective on microorganisms.

The controversial efficacy of vitamin E for human male infertility.

Vitamin E (VE) is major lipophilic chain-breaking antioxidant which protects tissue polyunsaturated fatty acids (PUFA) against peroxidation, a property that could be beneficial in the male reproductive physiology because the membranes of germ cells and spermatozoa are very sensitive to oxidation because of their high content of PUFA. Some of the available data on the efficacy of VE as an oral drug for male infertility or as an additive during in vitro manipulations of spermatozoa were reviewed here, observing that they are often contradictory, possibly because: (1) antioxidant therapy could be ineffective in certain studies not concentrated on men in whom oxidative stress is implicated as an infertility factor, and (2) the VE antioxidant therapy is a double-edged sword strictly depending on the dosage or the in vitro concentration of the vitamin. Thus, further laboratory and clinical studies with better-defined experimental conditions should be performed to establish the in vitro and in vivo efficacy of VE for human male infertility.

Toxicological evaluation of commercial mineral water bottled in polyethylene terephthalate: a cytogenetic approach with Allium cepa

The aim of this study was to ascertain the possible toxicological effects of chemicals released into mineral water packaged in polyethylene terephthalate (PET) bottles. Two commercial mineral waters, bottled both in PET and glass and stored under different conditions, were examined using the Allium cepa test. The influence of the water samples on macroscopic (root length, colour and form) and microscopic (root tip mitotic index, chromosome aberrations) parameters was examined. The water samples were analysed after: (A) controlled-conditio n storage (no direct light exposure and 18 ± 2°C), (B) storage at 40|°C for 10 days, in the dark (migration test in accordance with 82/711/EEC), and (C) exposure to sunlight and varying temperatures (18 ± 38°C, mean temperature 25 ± 38°C). The two water samples bottled in PET induced cytogenetic aberrations regardless of the storage conditions. These signs of toxicity were evident even only 8 weeks after bottling, which is well within the recommended expiry date. Storage conditions were very important, as is suggested by the finding that chromosomal aberrations were particularly apparent after exposure to direct sunlight. However, as plant systems are not considered as primary screening tools by current international guidelines for mammalian systems, extrapolation of the results from this test system to other systems and, eventually, to human beings should be based on results from a battery of assays covering various metabolic pathways.

The food contaminant semicarbazide acts as an endocrine disrupter: Evidence from an integrated in vivo/in vitro approach

Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75, 140mg/kg bw pro die during the juvenile period. Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM 40 and 75mg/kg and delayed at 140mg/kg, the latter effect probably due to the significantly decreased body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose-response was not evidenced. Testosterone catabolism was altered in a gender-related way, aromatase activity was increased in treated males at 75 and 140mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo-in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis.

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays.

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity. This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays. In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor alpha, with a lower potency but comparable efficacy to that of 17 beta-estradiol. Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F(2alpha,) and bradykinin than tissues obtained from estradiol valerate treated rats. The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F(2alpha,) and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 beta-estradiol. Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.

Purity control of some Chinese crude herbal drugs marketed in Italy.

The widespread use of herbal drugs, among which those coming from eastern Countries, has created a more compelling need for quality, a pre-requisite that can influence safety. In the present study, 10 Chinese crude herbal drugs marketed in Italy (Radix Ginseng, Radix Astragali, Rhizoma Coptidis, Rhizoma Atractylodis Macrocephalae, Radix Bupleuri, Radix Rehmanniae, Radix Paeoniae Alba, Pericarpium Citri Reticulatae, Radix Polygalae, Radix Salviae Miltiorrhizae) were analysed by the following purity assays: foreign matter, total ash, microbial and heavy metal contamination. Each herbal drug was purchased in Italy from three different sources: two Chinese firms and one Chinese herbal shop. Except for the heavy metal content, the tests were performed according to the European Pharmacopoeia. The presence of parasites was shown in two samples; moreover, level of ash (in three samples), lead content (in one sample) and total viable aerobic count (in one sample), were higher than the limits set by the European or Italian Pharmacopoeias. Our results, even if obtained from a small number of herbal drugs, show some purity issues and underline the importance of the quality control, particularly for this kind of products whose therapeutic value is not always demonstrated.

Interactions between Δ-9THC and capsaicin on isolated lamb bladder detrusor

A number of studies have demonstrated that capsaicin, a capsicum alkaloid, can affect isolated bladder tissue with either a relaxation or a contraction, depending on the species, by acting on VR1 receptors. In a previous work on isolated lamb detrusor, we demonstrated that capsaicin generally produces a relaxation of the tissue; this relaxation seems to be mediated by CGRP. Endogenous cannabinoids, such as anandamide, produce some of their actions by stimulating VR1 receptors and this seems to cause the release of peptides, e.g. CGRP. The aim of this work was to ascertain whether a cannabinoid, delta-9-tetrahydrocannabinol (delta-9THC), was able to interfere with the response of the isolated lamb detrusor to capsaicin. A-9THC, at concentrations between 1.6 x 10(-7) and 1.3 x 10(-6) M, displayed no activity on tissues. Instead, following delta-9THC, most of the tissues responded to capsaicin with a contraction that was abolished by atropine (9.0 x 10(-7) M). It has been reported that cannabinoids can inhibit the release of CGRP by stimulation of CB1 and CB2 cannabinoid receptors. Delta-9THC could act stimulating these receptors and thus inhibiting CGRP release and vesical relaxation. The muscle relaxing component removal could favour the contracting component, usually not active.

Tachykinin-independent activity of capsaicin on in-vitro lamb detrusor

The capsicum alkaloid capsaicin is an afferent fibre exciter. In the vesical bladder, capsaicin acts by releasing peptides stored in afferent fibres. The aim of this work was to verify the activity of capsaicin on in‐vitro lamb urinary bladder and to ascertain whether this alkaloid evokes peptide release. Capsaicin relaxed about 80 % of the lamb detrusor muscle preparations tested and contracted about 20%. Whereas neurokinin A and substance P antagonists, administered alone or together, left the contractile responses to capsaicin unchanged, atropine and tetrodotoxin totally inhibited contraction. Ruthenium red and indometacin abolished contractions and relaxation. The substance P and neurokinin A antagonists and the NO‐synthesis inhibitor Nω‐nitro‐l‐arginine methyl ester (L‐NAME) left relaxation unchanged; conversely, the calcitonin gene‐related peptide antagonist αh‐CGRP (8–37) abolished this response. These results suggest that capsaicin relaxes lamb detrusor muscle not through tachykinins but by releasing CGRP from afferent fibres. Our observation that indometacin blocks the capsaicin response in in‐vitro lamb urinary bladder also suggests a role of prostanoids.