Aluminium phosphide-induced genetic and oxidative damages in vitro: attenuation by Laurus nobilis L. leaf extract.

OBJECTIVE: The present investigation was undertaken to assess the protective effect of Laurus nobilis leaf extract (LNE) against aluminum phosphide (AIP)-induced genotoxic and oxidative damages stress in cultured human blood cells in the presence of a metabolic activator (S9 mix).
MATERIALS AND METHODS: Sister chromatid exchange (SCE) and chromosome aberration (CA) assays were used to assess AlP-induced genotoxicity and to establish the protective effects of LNE. In addition, we determined total antioxidant capacity (TAC) and total oxidative status (TOS) levels in AlP and LNE treated cultures for biomonitoring the oxidative alterations.
RESULTS: There was significant increases (P < 0.05) in both SCE and CA frequencies of cultures treated with AlP as compared to controls. Our results also showed that AlP (58 mg/l) caused oxidative stress by altering TAC and TOS levels. However, co-application of LNE (25, 50, 100 and 200 mg/l) and AlP resulted in decreases of SCE, CA rates and TOS level and increases of TAC level as compared to the group treated with AlP alone.
CONCLUSION: The preventive role of LNE in alleviating AlP-induced DNA and oxidative damages was indicated for the first time in the present study.

Introduction

Aluminium phosphide (AIP) is a colorless and flammable pesticide that widely used to control insects, weeds, and pathogens in crops, in forest and ornamental nurseries, and in wood products. Though it is widely used in agricultural and in other applications, AlP was considered toxic in various organs like liver, heart and kidney of mammals.[1] AIP produced phosphine gas (a highly toxic gas), a mitochondrial poison that could interfere with oxidative phosphorylation and protein synthesis when reacted with water or acids.[2] AlP induced oxidative stress in animals and humans.[3] Moreover, AlP was a suspected carcinogen and a known clastogen which had been shown to produce chromosome damage in agricultural workers.[45] On the other hand bay tree or daphne, Laurus nobilis (Lauraceae), is a hardy evergreen tree that grows wild or cultivated. This plant is used in folk medicine for stomachic and carminative remedies, as well as for the treatment of gastric diseases in many part of the world.[67] Moreover, recent experimental results indicated that LNE had exhibited antioxidant and antibacterial properties. Also the most significant inhibition of lipid peroxidation (LPO) was obtained with extracts of laurel bark.[8]

Since AIP was developed its toxic effects mainly via oxidative stress antioxidants or antioxidant like compounds can be used as most effective protectors against its harmful effects on humans and animals. Limited efforts were performed to explore protective agents to minimize AIP toxicity. In fact, some antioxidant natured compounds including glutathione,[5] ethanolamine lipids,[9] melatonin, sweet almond[10] and digoxin[11] were examined for chemoprevention of AlP toxicity. For this reason it is worth carrying out an experimental evaluation of the anti-oxidative (By TAC and TOS assays) and anti-genotoxic roles of LNE against AIP-induced oxidative and genotoxic damages (by SCE and CA tests) in human lymphocytes culture in vitro.

Materials and Methods

Experimental Design

Leaf samples for various extractions and processing experiments were collected from Turkey in May 2010. The extract was manufactured from the dried leaves of L. nobilis applying ethanol extraction procedure. The extract was dried in a desiccator and it was referred to as ethanol extract. It was diluted with 2% Tween-80 to desired concentrations and used for the experiments. Aluminum phosphide (Cas No 20859-73-8; AIP) was also obtained from the company Detia Degesch® (Germany). And all other chemicals were purchased from Sigma® (USA).

Human peripheral blood lymphocyte cultures were set up according to a slight modification of the protocol described by Evans and O’Riordan.[12] Heparinized blood samples were obtained by venipuncture from four healthy women volunteers. The heparinize blood (0.5ml) was cultured in 6 ml of culture medium (Chromosome Medium B, Biochrom® Leonorenstr. 2-6.D, Berlin) with 5μg/ml of phytohemagglutinin (Biochrom®). A various concentrations (25, 50, 100 and 200 mg/l) extracts of LNE and AlP (58 mg/l) were tested in blood cultures. The doses were selected according to previous reports.[1314] SCE and CA rates were assessed in peripheral lymphocytes in the presence of a supplemented liver fraction (S9 mix). The cultures without extracts and AlP were studied as control- group. Mitomycin C (10-7 M) was used as the positive control in SCE and CA assays. Likewise, ascorbic acid (10 μM) and hydrogen peroxide (25 μM) were also used as the positive controls in TAC and TOS analysis, respectively.

SCE Assay

With the aim of providing successive visualization of SCEs, 5-bromo-2’-deoxyuridine (Sigma®) was added at culture initation. The cultures were incubated in complete darkness for 72 hrs at 37 °C. Exactly 70 hrs and 30 min after beginning the incubations, demecolcine (Ndiacetyl- N-methylcolchicine, Sigma®) was added to the cultures. After hypotonic treatment, centrifugation, and resuspension, the cell suspension was dropped onto chilled, grease-free microscopic slides, air-dried, aged for three days, and then differentially stained for the inspection of the SCE rate according to fluorescence plus Giemsa (FPG) procedure. For each treatment condition, well-spread twenty five second division metaphases containing 42 - 46 chromosomes in each cell were scored, and the values obtained were calculated as SCEs per cell.[15]

CA assay

Two hours prior to harvesting, 0.1 mL of colchicine (0.2 mg/ mL, Sigma) was added to the culture flask. To prepare slides, 3–5 drops of the fixed cell suspension were dropped on a clean slide and air-dried. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. For each treatment, 30 well-spread metaphases were analyzed to detect the presence of chromosomal aberrations. Criteria to classify the different types of aberrations (chromatid or chromosome gap and chromatid or chromosome break) were in accordance with the recommendation of EHC (Environmental Health Criteria) 46 for environmental monitoring of human populations.[16]

TAC and TOS Analysis

The major advantage of TAC test is to measure the antioxidant capacity of all antioxidants in a biological sample and not just the antioxidant capacity of a single compound. In this test, antioxidants in the sample reduce dark blue-green colored ABTS radical to colorless reduced ABTS form. The change of absorbance at 660 nm is related with total antioxidant level of the sample. The assay is calibrated with a stable antioxidant standard solution which is traditionally named as Trolox Equivalent that is a vitamin E analog.[1718] Since the measurement of different oxidant molecules separately is not practical and their oxidant effects are additive, the total oxidant status (TOS) of a sample is measured and this is named total peroxide (TP), serum oxidation activity (SOA), reactive oxygen metabolites (ROM) or some other synonyms. In TOS assay performed here, oxidants present in the sample oxidize the ferrous ion–chelator complex to ferric ion. The oxidation reaction is prolonged by enhancer molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with chromogen in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micro molar hydrogen peroxide equivalent per liter (μmol H2O2 Equiv./L).[18] The automated TAC and TOS assays were carried out by commercially available kits (Rel Assay Diagnostics®, Turkey) on plasma samples of AlP and LNE treated cultures for 2 hour.

Statistic

The statistical analysis of experimental values was performed by one-way analysis of variance (ANOVA) and Fisher's LSD test using the S.P.S.S. 13.0 software. The level of 0.05 was regarded as indicative of statistical significance for all tests.

Results

The effects of AlP and LNE on the number SCE and CA in human whole blood cultures are shown in Figures 1 and 2, respectively. Mitomycin C (10-7M) caused significant increases of SCE (by 142.8%) and CA (by 250.2%) frequencies as compared to control group. Likewise, AlP (58 mg/L) caused significant increases of SCE and CA frequencies on human peripheral lymphocytes by 79.6% and 196.8%, respectively, when compared with the controls. However, the LNE at all applied concentrations (25, 50, 100 and 200 mg/l) did not indicate significant differences (P > 0.05) in the number of SCE or CA rates statistically. Furthermore, the positive effect of LNE was established on AlP-induced SCE and CA formations [Figures 3 and 4]. The incidences of these endpoints were decreased in comparison with AlP-treated group. Also, this situation was related as depending on concentrations of LNE.

Figure 1

Sister chromatid exchanges of human lymphocytes (n = 4) treated with AlP and LNE for 72h. Values are presented as mean ± S.D.; n = 4, means in the figure followed by different letter are significantly different at the (p < 0,05) level, Control-: negative control; Control+: positive control (MMC: mitomycin C (10-7M); AlP:58 mg/; LNE1: 25 mg/l L. nobilis ethanol extract; LNE2: 50 mg/l L. nobilis ethanol extract; LNE3: 100 mg/l L. nobilis ethanol extract; LNE4: 200 mg/l L. nobilis ethanol extract

Figure 2

Chromosome aberrations of human lymphocytes (n = 4) treated with AlP and LNE for 72h. (Abbreviations are as in Figure 1.)

Figure 3

A sample metaphase from 200 mg/l of LNE-treated culture

Figure 4

A sample metaphase from 58 mg/l of AlP-treated culture. (Arrows show sister chromatid exchange points)

The effects of AlP and LNE on TAC and TOS levels in human whole blood cultures are shown in Table. Significant decreases of TOS level (by 241.5%) and significant increases of TAC level (by 120.6%) was observed in the positive controls. Similarly, AlP caused significant increases of SCE (by 142.8%) and CA (by 250.2%) frequencies as compared to control group. The TAC and TOS analysis showed that AlP alone (at concentration of 58 mg/l) caused oxidative stress by decreasing of TAC level (by 65.1%) and increasing of TOS level (by 61.9%) as compared to control value. On the contrary, LNE showed positive effects on TAC level (at 50, 100 and 200 mg/l) without changing TOS level in relation with applied concentration. Moreover, three doses of LNE (at 50, 100 and 200 mg/l) significantly (P < 0.05) decreased the TOS level (between the range of 2.1%-28.8%) and significantly increased the TAC level (between the range of 9.8%-36.5%) when compared with the culture treated with AlP alone [Table 1].

Table 1

The levels of TAC and TOS in plasma samples of cultured human blood cells treated with LNE and AlP

Discussion

Our results clearly indicated that AIP induced genotoxic damage in human lymphoctes culture. The observed CA and SCE rates were found statistically higher than control- group. In accordance with our finding, Barbosa et al,[19] found a statistically significant increase of MN frequency in the bone marrow and spleen lymphocytes in mice. In an in vitro study on Hepa 1c1c7 cells, it was determined that the exposure to AIP caused elevation of 8-hydroxyguanine (8-OH-Gua) level which is a major pre-mutagenic lesion generated from reactive oxygen species (ROS).[13] On the contrary, the results for MN showed no significant differences between phosphine fumigators and control individuals.[20] Likewise, Kligerman et al.[21] investigated the cytogenetic effects of PH3 using SCE, CA and MN assays in cultured CD-1 mice splenocyte cells. They found no increase in any of the studied cytogenetic endpoints at any of the concentrations examined. But, it was also reported that fumigant applicators were exposed to PH(3) had significantly increased CA and SCE rates in lymphocytes.[22] Our findings also revealed that AIP lead to oxidative damage on cultured human blood cells. In this study, AIP caused increase of TOS level and decrease of TAC level as compared to control group. In parallel with this finding, it was suggested previously that PH(3)-induced mutagenic and cytotoxic effects were due to increased ROS levels, probably hydroxyl radicals, initiating oxidative damage.[13] In addition, Proudfoot,[23] suggested that PH(3) caused hydroxyl radical associated damage such as LPO by inhibiting catalase and peroxidase enzymes.

The present investigation was also determined that LNE protected against in vitro oxidative and genetic damages by AIP. O’Brien et al.[24] provided evidence that non-nutrient dietary constituents could act as significant bioactive compounds and several plant extracts. As a matter of fact, a very high level of antioxidant activity of L. nobilis diet was observed in bovine brain.[25] Likewise, Speroni et al.[6] found that oral administration of LNE to experimental animals were in good agreement with their antioxidant capacity, confirming the relationship between pharmacological efficacy and antiradical activity. Recent reports were also exhibited the neuroprotective, cardioprotective and antidiabetic properties of LNE due to its antioxidant content.[2627] Dall’Acqua et al.[28] determined the phytochemical composition L. nobilis leaves and found ten different flavonoid O-glycosides, one flavonoid C-glycoside, catechin, and cinnamtannin B1. All of these compounds were reported to have much or less antioxidant potentials.[28-30]

Our results demonstrated that LNE possessed significant protection against AlP-induced oxidative DNA damage, which was at least partly due to its antioxidant properties through scavenging free radicals to ameliorate oxidative stress. More importantly, LNE itself was safe and had no side effects. Furthermore, the chemo-protective and antigenotoxic potentials of LNE as observed in this study are may be useful in human pathological conditions.