Abstract

Pesticides are toxic chemicals used to control pests, plant diseases, or undesirable vegetation. Trifluralin (TRF), causing toxicity and mutagenicity, is a dinitroaniline class herbicide used to control undesirable vegetation in agriculture. Aloe vera is a widely used plant having rich gel content and a wide area of utilization. The present study aims to investigate the protective effect of Aloe vera on Drosophila melanogaster toxicity caused by TRF. For this purpose, life span, larval toxicity, and genotoxicity tests were performed on D. melanogaster administered with concentration of TRF (0.1 mM) and Aloe vera (2.5%, 5% and 10%). The results showed that life span was improved in the female and male populations when TRF and Aloe vera was administered (2.5%, 5% and 10%). Furthermore, the larval toxicity experiment showed that TRF decreased the survival rate of larvae; however, TRF and Aloe vera (10%) increased it at the highest concentration. According to the Comet Assay, TRF was found to cause an increase in the DNA damage; on the other hand, when D. melanogaster was administered with TRF and Aloe vera, the DNA damage decreased gradually in a dose-dependent manner. In conclusion, Aloe vera could be a good candidate to reduce the harmful effects of TRF, one of the most used herbicides.

Keywords:
Trifluralin; Aloe vera; life span; DNA damage; larval toxicity.

HIGHLIGHTS

• The longevity of the female and male Drosophila melanogaster is improved at doses given Aloe vera.

• Trifluralin has been observed to cause DNA damage, while Aloe vera has been shown to reduce the DNA damage.

• Trifluralin reduced larval survival rate, Aloe vera increased larval survival rate at high concentration.

INTRODUCTION

The use of modern agricultural techniques has been on the increase not only to enhance the productivity of agricultural products but also to obtain better products. Therefore, also the use of pesticides has increased day by day. Pesticides adversely affect human health and the environment as they persist in agricultural products and the environment for a long time [¹1 Tiryaki O, Canhilal R, Horuz S. The use of pesticides and their risks. Erciyes Uni. J.n Inst. Sci. Tech. 2010 Jun; 26 (2): 154-69,²2 Bolognesi C. Genotoxicity of pesticides: a review of human biomonitoring studies. Mutat. Res. 2003 Jun; 543:251-27.]. Herbicides are one of the pesticides used to kill or suppress the development of undesirable vegetation [³3 Kale PG, Petty BT, Jr Walker S, Ford JB, Dehkordi N, Tarasia S, et al. Mutagenicity testing of nine herbicides and pesticides currently used in agriculture. Environ. Mol. Mutagen.1995;25:148-53.]. Trifluralin (TRF; 2,6-dinitro-N, N-dipropyl-4-trifluoromethylaniline) is a Class 3 carcinogenic herbicide that falls into the class of dinitroaniline. According to the data from EPA (United States Environmental Protection Agency), it is reported that TRF has toxic effects above 0.5 ppm [⁴4 Sarıgol-Kiliç Z, Undeger-Bucurgat U. The apoptotic and anti-apoptotic effects of pendimethalin and trifluralin on A549 cells ın vitro. Turkish J. Pharm. Sci. 2018 Dec;15(3):364-9.] and is genotoxic in various experiments [⁵5 Li Y, Li C, Li B, Ma Z. Trifluralin residues in soils from main cotton fields of China and associated ecological risk. Chemosphere. 2021 Dec; 284, 131300.,⁶6 Fernandes TC, Mazzeo DEC, Marin-Morales MA. Mechanism of micronuclei formation in polyploidizated cells of Allium cepa exposed to trifluralin herbicide. Pestic. Biochem. Phys. 2007 July;88(3),252-9.,⁷7 Silva JM, de Brito Santos FL, Santos RV, de Oliveira Barreto E, Santos EL, Santana AEG, et al. Determination of genotoxic effect of trifluralin on Colossoma macropomum (Teleostei: Characidae: Serrasalminae, Cuvier, 1816) using a multibiomarker approach. Ecotoxicol. Environ. Cont. 2017 Apr;12(1),85-93.]. TRF has been used in agriculture since 1963. This herbicide is registered separately or in mixtures, and used in the following crops: Glycine max, Gossypium hirsutum, Arachis hypogaea, Phaseolus vulgaris, Allium sativum, Ricinus communis, Manihot esculenta, Solanum melongena, Daucus carota, Abelmoschus esculentus, Brassica oleracea, Brassica oleracea capitata, Brassica oleracea botrytis, Capsicum annuum, and ornamental plants [⁸8 Kilic ZS, Aydin S, Bucurgat UU, Basaran N. In vitro genotoxicity assessment of dinitroaniline herbicides pendimethalin and trifluralin. Food Chem. Toxicol. 2018 Mar; 113,90-8.]. TRF is greatly persistent in the environment and it can serve as a biotoxin and leads to genotoxicity in terrestrial organisms, including humans [⁹9 Capasso F, Borrelli F, Capasso R, Di Carlo G. Aloe and its therapeutic use. Phytother. Res. 1998 Dec;12(1):124-7.]. Furthermore it is an agent that endorses a cellular damage because of its direct action on the microtubules [¹⁰10 Lanka S. A review on Aloe vera the wonder medicinal plant. J. Drug Deliv. Ther. 2018 Oct; 8 (5-s): 94-9.]. The genotoxicity of TRF herbicide on Colossoma macropomum was verified by the micronucleus test and comet assay [¹¹11 Stanic S. Anti-genotoxic effect of Aloe vera gel on the mutagenic action of ethylmethane sulfonate. Arch. Biol. Sci. 2007 Jan; 59:223-6.]. TRF has caused the genotoxicity through the activation of oxidative stress pathway and chromosomal damage in Chinese hamster lung fibroblast (V79) cells [¹²12 Rani U, Singh M, Selwa KK. Aphrodisiac Effect of Aloe vera Gel Supplementation in Diet of Drosophila melanogaster Meigen. Indian J. Entomol. 2021 Nov;1-4.].

Aloe vera (L.) Burm.f. is a clear and gel-like substance obtained from A. vera leaf [¹³13 Oluwatoyin-Ayoola S, Oyinkansola-Ishola H. The growth performance and genotoxicity effect of dietary Aloe vera on Oreochromis niloticus juveniles. Aceh J. Animal Sci. 2020 Jun; 5 (2): 92-7.]. A. vera plant is known to contain active components such as vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids [¹⁴14 Akinboro A, Jimoh A. Antigenotoxic potential of gel extract of Aloe vera against Sodium azidegenotoxicity in Allium cepa cells. J. Med. Herbs Ethnomed. 2021 Feb;7,1-5.]. It is reported that A. vera, which has anticarcinogenic, antibacterial, antiviral, anti-inflammatory and antioxidant activities, reduces the genotoxic effect of D. melanogaster reduced genotoxicity after D. melanogaster had mutated with ethyl methanesulfonate (EMS) [¹⁵15 Dikilitas M, Kocyigit A. Analysis of DNA damage in organisms via “single cell gel electrophoresis” (technical note): comet assay method. Harran Uni. J. Fac. Agricul. 2010 May; 14 (2): 77-89.]. In a study conducted it is revealed that the gel supplementation increased its reproductive performance [¹⁶16 Carmona ER, Guecheva TN, Creus A, Marcos R. Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster. Environ. Mol. Mutagen. 2010 Mar; 52:165-9.] and enhances the productivity of Oreochromis niloticus [¹⁷17 Pasyukova EG, Vieira C, Mackay TFC. Deficiency mapping of quantitative trait loci affecting life span in Drosophila melanogaster. Genetics. 2000 Nov; 156:1129-46.]. This indicates that gel extract of A. vera includes phytochemical(s) that can be beneficial in the development of anticancer drug [¹⁸18 Cakir S, Sarikaya R. Effects of Some Organophosphate Insecticides on Percentage of Survival of Drosophila Melanogaster. Gazi Uni. J.Gazi Edu. Fac. 2004 Sep; 3(24): 71-80.].

Single-cell gel electrophoresis (SCGE) or Comet Assay method was used in our study to determine the effects of genotoxic agents on cells and to examine the damages that might occur in the DNA of cells [¹⁹19 YanMei D. Effects of aloe on the lifespan and SOD activity of Drosophila melanogaster. Medicinal Plant. 2011;(2)9:22-4.]. The simplicity of the Comet Assay and its use in all kinds of tissues/cells increases the applicability of the test [²⁰20 Sartiha V, Anilakumar KR, Khanum F. Antioxidant and antibacterial activity of Aloe vera gel extracts. Int. J. Pharm. Biol. Arch. 2010; 1(4): 376-84.]. Lifespan of the D. melanogaster is affected by genotoxic agents which is a quantitative feature of phenotypic variation in natural populations based on both genetic and environmental components [²¹21 Kızılet H, Uysal H. Ergin Induced Toxicity with Intake Chronic Zearalenone on the Life Span of Adult Drosophila. 2012 May; 43 (1): 1-5.]. D. melanogaster is a model organism preferred to examine the effects of chemicals with genotoxic effects [²²22 Uysal H, Semerdoken S. The assesment of longevity effects and larval toxicity of synthetic food dyes on Oregon R wild type of Drosophila Melanogaster. Kafkas Uni. J. Grad. School Nat. Applied Sci. 2011 July; 4(1):71-87.].

In this study, it was aimed to determine the protective effect of A. vera against genotoxicity in D. melanogaster given TRF for the first time. For this purpose; larval toxicity, life span analyses and comet assay of fly tissues were performed.

MATERIAL AND METHODS

Organism Used

In this study, we used Oregon-R wild strain of D. melanogasters in laboratory stock, which are genetically homozygous and non-mutant and have been used for many years at the Genetics Research Laboratory within Bartın University, Faculty of Science, Department of Biology.

Chemical Substances Used

The following chemicals were used: Trifluralin (2,6-Dinitro-N, N-dipropyl-4-trifluoromethylaniline, Cas No: 1582-09-8, Santa Cruz, USA), agar (Sigma Aldric, USA), diethyl ether (IsoLab, Germany), propionic acid (Merck, Germany), orthophosphoric acid (Merck, Germany), xylene (Sigma Aldric, USA), sodium chloride (IsoLab, Germany), phosphate buffered saline (Multicell), ethylenediaminetetraacetic acid (Sigma Aldric, Germany), sodium hydroxide (IsoLab, Germany), trizma base (Sigma Aldric, USA), low melting agarose (BioShop Canada Inc.), normal melting agarose (Peqlab), triton-X (Sigma Aldric, USA), dimethyl sulfoxide (Merck, Germany), ethidium bromide (Molecula, USA). A. vera extract was obtained in laboratory according to the methods of YanMei (2011) and Chandrashekara and Shakarad (2011).

Drosophila melanogaster third-stage larvae and adults were exposed to concentrations of 0.1 mM TRF and A. vera (2.5, 5 and 10%). TRF was dissolved in xylene. A. vera gel extract was obtained according to the method of Saritha et al [²³23 Olive PL, Banáth JP. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 2006 Feb;1(1):23-9.].

Life Span Experiment

For each experimental group, 15 individuals of the same age (1-3 days old) consisting of non-mated ♀♀ and ♂♂ flies were collected. During the experiment, D. melanogaster was fed in grouped culture flasks (Group 1: Control, group 2: Xylene, group 3: 0.1 mM TRF, group 4: 2.5% Aloe vera +0.1 mM TRF, group 5: 5% Aloe vera +0.1 mM TRF, group 6: 10% Aloe vera + 0.1 mM TRF) containing different concentrations of TRF and A. vera. During the experiment, food was refreshed once a week. The number of individuals was tracked on a daily basis and this continued until the last individual died. The experiments were repeated twice [²⁴24 Pilinskaia MA. Evaluation of the cytogenetic effect of the herbicide treflan and of a number of its metabolites on mammalian somatic cells. Tsitol. Genet. 1987 Mar; 21 (2):131-5.].

Larval Toxicity

Larval toxicity experiments were performed using the 3rd stage (72±4 hours) larvae of D. melanogaster. Larvae were obtained by crossing 10 ♂♂ individuals with 10 ♀♀ individuals in culture medium containing standard growth medium. After mating of flies, the 3rd stage larvae were collected and 50 larvae were added to each group (Group 1: Control, group 2: Xylene, group 3: 0.1 mM TRF, group 4: 2.5% Aloe vera +0.1 mM TRF, group 5: 5% Aloe vera +0.1 mM TRF, group 6: 10% Aloe vera + 0.1 mM TRF) culture flask. Larvae development was monitored on a daily basis and the number of flies were noted by sex for 15 days. The experiment was repeated twice [²⁵25 Nehez M, Paldy A, Selypes A, Korosfalvi M, Lorinczi IL, Berencsi G. The mutagenic effect of trifluralin-containing herbicide on mouse germ cells in vivo. Ecotoxicol. Environ. Saf. 1980 Sep; 4(3):263-6.].

Comet Assay

Comet assay was performed on groups of homogenized fly tissues (~10,000 cells per slide). (Group 1: Control, group 2: Xylene, group 3: 0.1 mM TRF, group 4: 2.5% Aloe vera +0.1 mM TRF, group 5: 5% Aloe vera +0.1 mM TRF, group 6: 10% Aloe vera + 0.1 mM TRF) that were administered with TRF and Aloe vera extract for 15 days in order to determine the damage that occurred in DNA. According to the method of Dhawan et al. (2009), the fly samples were decomposed in HBSS (containing 20 mM EDTA and 10% DMSO) solution and the cells were suspended in 0.5% low melting agarose (LMA) [²⁶26 Dhawan A, Bajpayee MM, Pandey AK, Parmar D. Protocol for the single cell gel electrophoresis/comet assay for rapid genotoxicity assessment. Sigma. 2003; 1077(1), 1-10.]. It was added on slides coated with 1% normal melting agarose (NMA) and allowed to freeze. The preparations were kept in the lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, pH adjusted to 10, and 1% Triton X-100, 10% fresh DMSO to be added) at 4°C for 1 hour. The preparations were kept in electrophoresis buffer (10N NaOH, 200mM EDTA, pH> 13.0) at 4°C for 15 minutes. Alkaline electrophoresis was applied at 24 V and 300 mA for 40 minutes. The preparations were neutralized in 0.4 M Tris buffer (pH 7.5) for 5 minutes. The preparations were then stained with 100 μL Red Safe (10 μL/mL) and fluorescence microscopy (Zeiss, Germany) was used to examine them [²⁷27 Bozari S, Aksakal O. Application of random amplified polymorphic DNA (RAPD) to detect genotoxic effect of trifluralin on maize (Zea mays). Drug Chem.Toxicol. 2013; 36(2), 163-9.]. While examining the obtained images, damage records from 0 to 4 were noted in 100 randomly selected cells (0 = undamaged, 4 = highly damaged) [²⁸28 De Oliveira B, Pereira LC, Pazin M, Franco Bernanrdes MF, Dorta DJ. Do trifluralin and tebuthiuron impair isolated rat liver mitochondria? Pestic. Biochem. Phys. 2020; 163:175-84.].

Statistical Analysis

In the study, the Duncan's post-hoc test was applied to evaluate the data obtained from female and male life span and larval toxicity experiments and to evaluate the significance of the difference between the averages by one-way analysis of variance (One-Way ANOVA) (SPSS 20) in intergroup evaluations. The statistical significance of the average was considered as p<0.05.

RESULTS

When the data on life span were examined, it was observed that the average life span of male and female individuals increased compared to the control groups (Table 2). It was observed that the life span was reduced in the groups administered with TRF whereas the life span was improved in the TRF + A. vera (2.5-5-10%) groups. When female and male individuals were compared to each other, it was seen that male individuals lived longer than female individuals in control and all experimental groups.

When the effect of TRF and A. vera on the survival rate of D. melanogaster was examined, it was observed that the survival rate of the larvae was reduced at the specified concentration of TRF. In the control group, the 3^rd stage larvae were obtained at 86±1.00% concentration. In the xylene group, the 3^rd stage larvae were obtained at 18±1.50% concentration, in direct proportion, due to the high proportion of xylene. At TRF+5% Aloe vera concentration, the proportion of larvae reaching the 3^rd stage decreased. At the TRF+10% Aloe vera concentration, the proportion of larvae reaching the 3^rd stage increased. At TRF concentration and TRF+2.5% A. vera concentration, the proportion of larvae reaching the 3^rd stage decreased (Table 1). The rate of D. melonagaster's transition from pupa to adult was analyzed among all experimental groups. The rate of those that matured is 86% in control group, 82% in the TRF experimental group, 63% in the TRF+2.5% A. vera experimental group, 84% in the TRF+5% Aloe vera experimental group, and 97% in the TRF+10% A. vera experimental group (Table 1).

When examining the Comet images, the damage in tail length of 100 randomly selected cells was rated from 0 to 4. As a result, it was observed that TRF caused an increase in DNA damage in female and male flies whereas the DNA damage decreased, depending on the dose, at the concentrations of TRF+2.5% A. vera, TRF+5% Aloe vera and TRF+10% A. vera (Figure 1).

Figure 1
(A) Classified comet image showing DNA damage. (B-C) Graphical representation of the damage caused by exposure of adult female (B) and male (C) D.melanogaster populations to different concentrations of TRF (0.1 mM) and Aloe vera (2.5% - 5% and 10%) for 15 days (Group 1: Control, group 2: Xylene, group 3: 0.1 mM TRF, group 4: 2.5% Aloe vera +0.1 mM TRF, group 5: 5% Aloe vera +0.1 mM TRF, group 6: 10% Aloe vera + 0.1 mM TRF). (p<0.05)

DISCUSSION

In our study, the protective effect of A. vera extract against the toxic and genotoxic effects of TRF (0.1 mM) was investigated. In a study conducted on the genotoxic effect of TRF was investigated in lymphocyte cells of humans and bone marrow cells of mice by using chromosome aberration technique and it was concluded that the use of TRF in an amount that is genetically hazardous should be regulated [²⁹29 Kaya B, Marcos R, Yanikoglu A, Creus A. Evaluation of the genotoxicity of four herbicides in the wing spot test of Drosophila melanogaster using two different strains. Mutat. Res. 2004 Jan; 557:53-62.]. It was seen that Olitref herbicide containing 26% TRF had mutagenic effects on the reproductive cells of mice [³⁰30 Konen S, Cavaş T. Genotoxicity testing of the herbicide trifluralin and its commercial formulation treflan using the piscine micronucleus test. Environ. Mol. Mutagen. 2008 Jul; 49:434-8.]. TRF’s genotoxic potential has been shown on maize (Zea mays) by using the random amplified polymorphic DNA (RAPD) technique [³¹31 Sacan O, Akev N, Yanardag R. In vitro inhibitory effect of Aloe vera (L.) Burm. f. leaf extracts on activity of some enzymes and antioxidant activity. Indian J. Biochem. Biophys. 2017 Feb; 54:82-9.]. It was also observed that TRF affected mitochondrial respiration at high concentrations [³²32 Kumar Gupta V, Kumar A, De Lourdes Pereira M, Jamal Siddiqi N, Sharma B. Anti-inflammatory and antioxidative potential of Aloe vera on the cartap and malathion mediated toxicity in Wistar rats. Int. J. Environ. Res. Public Health. 2020 Jul; 17;17(14):5177.]. Similarly, in a study regarding genotoxicity, immunotoxicity and reproductive toxicity of TRF, it was reported that TRF was genotoxic in line with the SMART test on D. melanogaster as is the case in our study [³³33 Chandrashekara KT, Shakarad MN. Aloe vera or resveratrol supplementation in larval diet delays adult aging in the fruit fly, Drosophila melanogaster. J. Gerontol. A Biol. Sci. 2011 Sep; 66: 965-71.]. It was observed that the survival rate of larvae decreased at the specified concentration of TRF in the larval toxicity study within the scope of our study.

The genotoxic effects of treflan, a commercial form of TRF, on Oreochromis niloticus, an economically important fish species, were studied by using the micronucleus test and morphological nucleus irregularity analysis and it was shown that ascorbic acid had a decreasing effect on the genotoxicity caused by TRF and Treflan [³⁴34 Yun N, Lee CH, Lee SM. Protective effect of Aloe vera on polymicrobial sepsis in mice. Food Chem. Toxicol. 2009 Jun; 47(6):1341-8.]. It was found out in this study conducted coherently with these studies that A. vera extract, a natural antioxidant source, [³⁵35 Behmanesh MA, Najafzadehvarzi H, Poormoosavi SM. Protective Effect of Aloe vera Extract against Bisphenol A Induced Testicular Toxicity in Wistar Rats. Cell J. (Yakhteh). 2018 Mar; 20(2),278.] It was seen that A. vera leaves had a curative effect against malathion toxicity in rats [³⁶36 Ayaz A, Yurttagul M. Toxic elements in foods II. 2nd edition: 727, Ankara, 2008. 37 s.]. It was found that resveratrol and A. vera administration to D. melanogaster increased the life span [³⁷37 Toros S, Maden S, Sozeri S. Agricultural control methods and drugs. 4th edition, Ankara Uni. Fac. Agric. Public. No:1520, Ankara, 2001. 417 s.]. In this study, it was found that the life span of the experimental groups administered with A. vera and TRF (0.1 mM) increased.

Protective effect of A. vera can be a therapeutic agent potentially for the clinical treatment of sepsis on polymicrobial sepsis in mice [³⁸38 Aydinoglu H, Dursun HY, Bayraktar L. “Plant Protection Products”, Ministry of Agriculture and Rural Affairs, General Directorate of Control, Ankara, 2002. 155-365 p.]. According to another study, A. vera gel extract can counteract the damaging effects of BPA on the reproductive system of rats and protects rats’ testes against bisphenol-a (BPA)-induced toxicity [³⁹39 Fernandes TC, Pizano MA, Marin-Morales MA. Characterization, modes of action and effects of trifluralin: a review. IntechOpen. 2013.]. It is seen in out study that A. vera which is known to have a protective effect counteracts harmful effects of TRF. The rate of larvae reaching the 3^rd stage and the rate of maturity increased at TRF+A. vera (5-10%) concentration whereas they decreased in the TRF+A.vera (2.5%) group. According to the Comet Assay, it was observed that TRF increased the DNA damage on D. melanogaster, and that, when used with TRF+A. vera (2.5, 5 and 10%), the DNA damage decreased.

As a result, it is seen that A. vera can be used as a preservative alongside TRF and can reduce the harmful effects of TRF, and it is considered that A. vera gel has a significant role for human health and vitality and our study contributes to the literature.

REFERENCES

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