Effects of Chlorophenoxy Herbicides and Their Main Transformation Products on DNA...

Research ArticleEffects of Chlorophenoxy Herbicides andTheir Main Transformation Products on DNA Damage andAcetylcholinesterase Activity

Sofia Benfeito12 Tiago Silva2 Jorge Garrido12 Paula B Andrade3 M J Sottomayor2

Fernanda Borges2 and E Manuela Garrido12

1 Departamento de Engenharia Quımica Instituto Superior de Engenharia do Porto (ISEP) Instituto Politecnico Porto4200-072 Porto Portugal

2 CIQDepartamento de Quımica e Bioquımica Faculdade de Ciencias Universidade do Porto 4169-007 Porto Portugal3 REQUIMTELaboratorio de Farmacognosia Departamento de Quımica Faculdade de Farmacia Universidade do Porto4050-313 Porto Portugal

Correspondence should be addressed to Jorge Garrido jjgisepipppt

Received 28 November 2013 Accepted 4 February 2014 Published 27 March 2014

Academic Editor Peter P Egeghy

Copyright copy 2014 Sofia Benfeito et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Persistent pesticide transformation products (TPs) are increasingly being detected among different environmental compartmentsincluding groundwater and surface water However there is no sufficient experimental data on their toxicological potential to assessthe risk associated with TPs even if their occurrence is known In this study the interaction of chlorophenoxy herbicides (MCPAmecoprop 24-D and dichlorprop) and their main transformation products with calf thymus DNA by UV-visible absorptionspectroscopy has been assessed Additionally the toxicity of the chlorophenoxy herbicides and TPs was also assessed evaluatingthe inhibition of acetylcholinesterase activity On the basis of the results found it seems that AChE is not the main target ofchlorophenoxy herbicides and their TPs However the results found showed that the transformation products displayed a higherinhibitory activity when compared with the parent herbicides The results obtained in the DNA interaction studies showed ingeneral a slight effect on the stability of the double helix However the data found for 4-chloro-2-methyl-6-nitrophenol suggestthat this transformation product can interact with DNA through a noncovalent mode

1 Introduction

World population is expected to grow by over a third between2009 and 2050 The projections show that feeding a worldpopulation of 91 billion people in 2050 would require raisingoverall food production by about 70 percent between 200507and 2050 [1] The rate of growth in world demand for agri-cultural goods may lead to increased use of chemical controlproducts since these substances can contribute considerablyto increasing yields and improve farm revenues

Pesticides a broad group of biologically active com-pounds used for pest management are among the mostwidely used chemicals in the world and also among the mostdangerous to environmental and human health The impactof pesticide molecules on the environment depends on

several factors their toxicity their bioaccumulation and long-term effects their transport between different compartmentsand their persistence in the environment [2] There is alsoincreasing interest in their transformation products (TPs)since these by-products can play a significant role in definingthe impact of pesticides on both human health and the natu-ral ecosystemsThe formation and environmental presence ofTPs thus add further complexity to chemical risk assessmentTPs may contribute significantly to the risk posed by theparent compound (a) if they are formed with a high yield (b)if they are more persistent or more mobile than the parentcompounds or (c) if they have higher toxicity [3]

The adverse health effects of pesticide use have longbeen established with links to neurologic and endocrine(hormone) system disorders birth defects cancer and other

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 709036 10 pageshttpdxdoiorg1011552014709036

2 BioMed Research International

diseases [4 5] Although in general pesticide TPs showlower toxicity to biota than the parent compounds in somecases TPs are more toxic and represent a greater risk to theenvironment and health than the parent molecules [3 6]Therefore toxicological evaluation of pesticide TPs is consid-ered to be an emerging issue

Acetylcholinesterase enzyme (AChE) is very importantfor the central nerve system of humans and insects AChE isan enzyme that degrades through its hydrolytic activity theneurotransmitter acetylcholine and is critically important forthe regulation of neurotransmission at synapses in all areasof the nervous system [7] If AChE activity is blocked acetyl-choline accumulates at cholinergic receptor sites therebyexcessively stimulating the cholinergic receptors This canlead to various clinical complications including fibrillationleading ultimately to death [7] So large-scale inactivation ofAChE has lethal consequences for any organism with a ner-vous system It is well demonstrated that AChE is inhibitedby neurotoxins namely organophosphates and carbamatepesticides and many other compounds [8 9] In fact recentreports have also described that some fungicides and herbi-cides used in agrochemicals can also damage AChE [10 11]The evaluation of AchE inhibitory activity can help to deter-mine the toxicity levels of a diversity of net pesticides andtheir degradation products

Besides the enzymatic damage in the biologic systemspesticides have been shown to have the capacity of interactingwith the DNA Chemicals that interact with DNA can causedirect damage by covalent modifications such as adducts orstrand breaks or can perturb DNA and chromatin functionby noncovalent binding [12ndash14]Thus the preliminary evalu-ation of DNA damage is considered to be of high significancefor the study of the putative interactions of pesticides andtheir TPs with DNA and for the screening of their mutagenicproperties

Chlorophenoxy herbicides are used worldwide as plantgrowth regulators for agricultural and nonagricultural pur-poses [15 16] These herbicides can be easily transferred tosurface and ground waters due to their polar nature and rela-tively good solubility which increase the risks of contamina-tion and consequently the environmental damage [17] Basedon epidemiologic studies phenoxy acid herbicides have beenclassified by the International Agency for Research onCancer(IARC) as possibly carcinogenic to humans (category 2B)[18] The most commonly reported transformation interme-diates of phenoxy acid degradation are the correspondingchlorophenols and their nitroderivatives the latter beingenvironmentally more persistent than the parent molecules[19 20] In fact the key metabolite in the degradation ofphenoxy acids is the corresponding chlorophenol that resultsfrom cleavage of the ether bond in the parent compoundThe occurrence of nitrophenols as transformation productsis a consequence of a photochemical nitration process ofthe corresponding phenols [19] Due to the environmentaloccurrence of these compounds their risk assessment is veryrelevant because the nitration of chlorophenols reduces theiracute toxicity but the nitroderivatives could have moremarked long-term effects associated with their genotoxicity[20 21]

The aim of this work was to evaluate the interac-tion of four chlorophenoxy herbicides (MCPA meco-prop 24-D and dichlorprop) as well as their maintransformation products (24-dichlorophenol 4-chloro-2-methylphenol 24-dichloro-6-nitrophenol and 4-chloro-2-methyl-6-nitrophenol) with calf thymus DNA by UV-visibleabsorption spectroscopy (Figure 1) Thermodynamic param-eters of DNA thermal denaturation in solutions containingthe herbicides and their TPs have been evaluated to interpretthe mode of interaction Furthermore the toxicity of thechlorophenoxy herbicides and TPs was also assessed evalu-ating their inhibitory activity towards acetylcholinesterase

2 Experimental

21 Chemicals and Reagents The phenoxy acid herbicidesand the transformation products (except 4-chloro-2-methyl-6-nitrophenol that was obtained by synthesis) as well asNaCl MgCl

2sdot6H2O Na

2HPO4and NaH

2PO4were supplied

by Sigma-Aldrich (Sintra Portugal) and used without furtherpurification Bovine serum albumin (BSA) 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) acetylthiocholine iodide (ATCI)galantamine AChE (CAS 9000-81-1 EC 232-559-3) fromelectric eel (typeVI-s lyophilized powder) Tris-HCl and calfthymus DNA as Type I calf thymus DNA sodium salt (pro-tein content lt 3) were also purchased from Sigma-AldrichUltrapure (Type 1) water (Millipore Milli Q Gradient) wasused throughout the experiments

The following buffers were used in the acetyl-cholinesterase activity assay buffer A 50mM Tris-HClpH 8 buffer B 50mM Tris-HCl pH 8 containing 01BSA and buffer C 50mM Tris-HCl pH 8 containing01M NaCl and 002M MgCl

2sdot6H2O Acetylcholinesterase

from Electrophorus electricus (electric eel) (type VI-slyophilized powder 425Umg 687mgprotein) was used inthe enzymatic assay The lyophilized enzyme was dissolvedin buffer A to make 1000UmL stock solution and furtherdiluted with buffer B to get 044UmL of enzyme in themicroplate well

A stock solution (10 times 10minus3mol dmminus3) of DNA wasprepared by dissolving an appropriate amount of DNAin 005mol dmminus3 phosphate buffer solution (pH 72 ionicstrength adjusted to 01mol dmminus3 withNaCl) Stock solutions(20 times 10minus4mol dmminus3) of herbicides were prepared by disso-lution of a suitable quantity in ultrapure water

The UV absorbance measurements were performed inpH 72 phosphate buffer solutions with ionic strength001mol dmminus3 Working solutions of DNA (70 times10minus5mol dmminus3) herbicides (50 times 10minus5mol dmminus3 or 10times 10minus4mol dmminus3) and DNAherbicides were prepared bysimple dilution of the appropriate amounts of the stocksolutions in phosphate buffer and ultrapure water

The concentration of DNA solutions expressed in molesof base pairs was determined at 20∘C by UV spectroscopyat 260 nm using a molar absorption coefficient 120576

260=

13200molminus1 dm3 cmminus1 [22] A ratio of absorbance at 260 nmto that at 280 nm (119860

260119860280

) greater than 18 indicatedthat DNA was sufficiently pure and free from protein [23]

BioMed Research International 3

ClCl

O

R

OH

OCH3

MCPA R = H

Mecoprop R = CH3

(a)

O

R

OH

O

Cl

Cl

24-D R = H

Dichlorprop R = CH3

(b)

OH

R

Cl

R = CH3

R = Cl

4-Chloro-2-methylphenol

24-Dichlorophenol

(c)

OH

R

Cl NO2

R = CH3

R = Cl

4-Chloro-2-methyl-6-nitrophenol

24-Dichloro-6-nitrophenol

(d)

Figure 1 Chemical structures of the chlorophenoxy herbicides and their main environmental transformation products (a) MCPA andmecoprop (b) 24-D and dichlorprop (c) 4-chloro-2-methylphenol and 24-dichlorophenol and (d) 4-chloro-2-methyl-6-nitrophenol and24-dichloro-6-nitrophenol

Control experiments under the same conditions and initialconcentrations of herbicides were carried out in parallel forcomparison All the solutions were stored in the refrigeratorat 4∘C

22 Synthesis of 4-Chloro-2-methyl-6-nitrophenol The syn-thetic procedure was adapted from the literature withslight modifications [24 25] To a solution of 4-chloro-2-methylphenol (03 g 22mmol) in CH

2Cl2(30mL) NaNO

2

(03 g 40mmol) Al(HSO4)3(04 g 14mmol) and wet SiO

2

(50 ww) (044 g) were added The resulting mixture wasstirred and the reaction was completed after 90min Thecrude product was extracted with CH

2Cl2(2 times 15mL) The

organic phases were combined washed with water (2 times15mL) dried over anhydrous Na

2SO4and evaporated under

reduced pressure The nitrated product was purified on asilica gel column using CH

2Cl2MeOH (9 1 and 8 2 ratio

(vv)) as eluting system The fractions containing the desiredcompound were collected and the solvent evaporated in arotary evaporator1HNMR (400MHz CH

3OH) 120575 = 230 (3H s CH

3) 752

(1H d 119869 = 26Hz H (1)) 792 (1H d 119869 = 26Hz H (2))MSEI119898119911 189 (28) 187 (M+∙ 100)

23 Evaluation of Acetylcholinesterase Inhibitory ActivityAcetylcholinesterase activity was determined spectropho-tometrically using a 96-well Multiskan Ascent microplate

reader (Thermo Electron Corporation) based on Ellmanrsquosmethod according to a previously described procedure [26]In each well the mixture consisted of 25 120583L of 15mM ATCIin water 125 120583L of 3mM DTNB in buffer C 50 120583L of bufferB and 25 120583L of 10mM solution of each compound understudy (dissolved in a solution of 10 methanol in buffer A)The absorbance was measured at 405 nm After this step25 120583L of AChE (044UmL) was added to each well and theabsorbance was measured again at the same wavelength Therates of the reactions were calculated by Ascent Softwareversion 26 (Thermo Labsystems Oy)The rate of the reactionbefore adding the enzyme was subtracted from that obtainedafter enzyme addition in order to correct for spontaneoushydrolysis of substrateThe enzymatic activity was calculatedby comparing the rates of the samples with the control (10methanol in buffer A) Galantamine a reversible competitiveinhibitor of acetylcholinesterase was used as reference com-pound [27]The results are expressed as the mean percentageof inhibition obtained from three separate experiments

24 UV Spectroscopy Experiments Theabsorption spectra aswell as the UV melting curves were recorded on a hermeticquartz cell with a 1 cm path length using an Agilent 8453UV-Vis spectroscopy system equipped with a thermostatic cellholderThe temperature of the samples was controlled using aJulabo F25HP thermostatic bathTheUV absorption spectra


of herbicides and DNAherbicides solutions were acquiredin the wavelength range of 200ndash400 nm at 20∘C For DNAmelting studies the temperature of the cell was changed from20 to 95∘C with a heating rate of 1∘Cminminus1

The fraction of melted base pairs 120579 at each temper-ature has been calculated from the curves of absorbanceversus temperature as described elsewhere [28] The meltingtemperature 119879

119898 defined as the temperature at which half

of the amount of DNA is denatured was determined byinterpolation in the curves 120579 = 119891(119879) for 120579 = 05 [28] Thehyperchromicity at 260 nm119867

260 was calculated as described

elsewhere [28]

25 Thermodynamic Parameters of DNA Thermal Denat-uration The thermodynamic parameters of DNA thermaldenaturation were obtained from the denaturation curves bytwo different methods based on the vanrsquot Hoff equation [28]Both methods assume that denaturation is a two-state transi-tion and the values of enthalpy and entropy are not dependenton temperature

Briefly the first method is based on the dependence of theequilibrium constant 119870 of DNA denaturation on tempera-ture 119879 The value of 119870 at each temperature can be expressedas a function of the broken base pairs fraction 120579 The vanrsquotHoff denaturation enthalpy and entropy values were obtainedby linear regression of minus ln119870 versus 1119879 and fitting the datato values of 120579 from 025 to 075 [28] In the second methodthe vanrsquot Hoff denaturation enthalpy and entropy valueswere calculated using the peak height maxima obtainedfrom the derivative melting curves (119889120579119889119879)max [28]

Values of Δ119867ovH and Δ119878ovH obtained from both methods

differed by less than plusmn1 Thus the values given correspondto the average calculated from the values obtained using bothmethods

3 Results and Discussion

31 Evaluation of Acetylcholinesterase Inhibitory ActivityThe inhibition of peripheral enzymes such as acetyl-cholinesterase provides a convenient and nondestructivemean of monitoring exposure to pesticides and their trans-formation products and has been widely implemented byregulatory agencies [29] Monitoring of AChE inhibition iswidely used as a biomarker of organophosphorus and carba-mate pesticide exposure either in aquatic or terrestrial envi-ronments [29] A number of important contaminants otherthan these pesticide groups have recently been shown tohave anticholinesterase properties namely heavy metals andherbicides [29]

The in vitro screening assay most frequently used toevaluate the inhibitory activity of a contaminant towardsAChE is based on Ellmanrsquos method [30] Accordingly thechlorophenoxy herbicides and their transformation productswere evaluated for their inhibitory activities toward AChEin comparison with galantamine as reference drug Theanticholinesterase activities are summarized in Table 1 Nosignificant inhibitory activities towards AchE were found

Table 1 Acetylcholinesterase inhibitory activity of chlorophenoxyherbicides and its transformation products

Compound inhibition plusmn SDa

MCPA 129 plusmn 15

Mecoprop Inactive4-Chloro-2-methylphenol 190 plusmn 07

4-Chloro-2-methyl-6-nitrophenol 284 plusmn 59

24-D 39 plusmn 003

Dichlorprop Inactive24-Dichlorophenol 229 plusmn 16

24-Dichloro-6-nitrophenol 358 plusmn 42

Galantamine 957 plusmn 005

aResults are expressed as mean plusmn standard deviation of three independentdeterminations

for the chlorophenoxy herbicides under study when com-pared with the reference drug However the results foundrevealed that the transformation products displayed a rathernoticeable inhibitory activity when comparedwith the parentherbicides The data obtained show that the nitroderivativesexhibit the highest inhibitory activity among all the testedcompounds Actually the inhibitory activity of the com-pounds depends on the type and number of substituentsattached to the aromatic ringThese results reinforce the ideathat a particular attention should be placed on assessing theeffect of neurotoxicity of pesticides as in some cases theseeffects may only be observed after the formation of transfor-mation products which arise from environmental degrada-tion

32 Evaluation of Pesticides-DNA Interactions DNA is foundin cells and usually looks like a right-handed double helixThe two chains (strands) of the double helix are connectedby hydrogen bonds Small ligandmolecules can bind to DNAand artificially alter andor inhibit the functioning of DNAIn general four types of reversible binding modes can occurbetween molecules and double-helical DNA (i) electrostaticattractions with the anionic sugar-phosphate backbone ofDNA (ii) interactions with the major and minor grooves(iii) intercalation between base pairs via the DNA major andminor grooves and (iv) a threading intercalation mode [14]Depending on structural features of both the molecule andDNA somemolecules can present simultaneously more thana single interaction mode with DNA [14]

The mechanism of interactions between small moleculesand DNA is still unclear Thus various techniques have beenused to study the binding of small molecules with DNA withUV-visible absorption spectroscopy being the simplest andmost commonly employed instrumental technique

321 UV Absorption Spectra Some pesticides andor itsenvironmental transformation products have the capacity tointeract and damage the structure of the DNA a processthat often has a toxic outcome for human health Thus thedetermination of the specific type of structural DNA damagecan be viewed as a biomarker of damage exposure [31]


200 220 240 260 280 300 320 340000

010

020

030

040

050

060

070

080Ab

sorb

ance

Wavelength (nm)

MecopropMCPA

Dichlorprop24-D

4-Chloro-2-methylphenol4-Chloro-2ndashmethyl-6-nitrophenol

24-Dichlorophenol24-Dichloro-6-nitrophenol

(a)

200 220 240 260 280 300 320 340000

020

040

060

080

100

120

140

160

Abso

rban

ce

Wavelength (nm)MecopropMCPA4-Chloro-2-methylphenol4-Chloro-2ndashmethyl-6-nitrophenolDichlorprop24-D24-Dichlorophenol24-Dichloro-6-nitrophenol

(b)

Figure 2 UV-Vis absorption spectra of (a) 50 times 10minus5mol dmminus3 and (b) 10 times 10minus4mol dmminus3 of chlorophenoxy herbicides and TPs standardsolutions in pH 72 phosphate buffer electrolyte

The pesticide-DNA interaction can be efficiently detected byUV-Vis absorption spectroscopy bymeasuring the changes inthe absorption properties of the pesticide or theDNA [32 33]The magnitude of the shift of the position of the absorptionbands could be interpreted as an indication of the strength ofthe DNA-pesticide interaction [33]

The UV absorption spectra of chlorophenoxy herbicidesat two different concentrations in the absence of DNAare presented in Figure 2 Except for the nitroderivativeswhere only two bands are seen all the other studied com-pounds exhibit three absorption bands The weaker bandsin the 270ndash300 nm range correspond to 120587 rarr 120587lowast and119899 rarr 120587

lowast transitions that are characteristic of benzene andits substituted derivatives the band corresponding to themaximum absorption occurring below 240 nm is alsorelated to the existence of an aromatic system [34 35] Whennonbonding pair substituents (such NO

2) are present on the

benzene ring the absorptions are shifted substantially tolonger wavelengths and the fine structure of the B-band isseriously diminished or wholly eliminated because of 119899-120587conjugation [34]These changes can justify the occurrence ofa single band in the 270ndash300 nm range for nitroderivatives

Figure 3 shows the UV-visible absorption spectraobtained for DNA in the absence or presence ofchlorophenoxy herbicides and their transformation products(DNAherbicides and DNATPs) The UV-Vis absorptionspectrum of DNA exhibits a broad band (200ndash340 nm) in theUV region with amaximum absorption at 260 nm (Figure 3)This maximum is a consequence of the chromophoric

groups in purine and pyrimidine moieties responsiblefor the electronic transitions [32 33] Spectra registeredfor DNAherbicides and DNATPs solutions exhibit theabsorption bands common to both DNA and each of theherbicides or TPs under study (Figure 3) Except for thenitroderivatives for both concentrations tested only slightvariations are observed in the peak position and absorptionintensity of the bands for DNAherbicides solutionscompared to DNA

To evaluate the effect of chlorophenoxy herbicides andtransformation products on the UV spectrum of DNA thespectra of DNAherbicides and DNATPs solutions werecompared with the sum of the individual spectra of DNA andherbicide or TP at the same concentration (Figure 4) Exceptfor the nitroderivatives the UV spectra of DNAherbicidesand DNATPs solutions present only minor changes inthe absorption intensity of the bands relative to the sumof the spectra of DNA and each of the herbicides and TPs(Figure 4(a)) Since no significant variations are observedone can assume that the interaction of these herbicides andTPs withDNAbase pairs is not strong since nomarked effectwas observed in the structure of the DNA molecule On thecontrary for the nitroderivatives (24-dichloro-6-nitrophenoland 4-chloro-2-methyl-6-nitrophenol) (Figure 4(b)) ameaningful bathochromic shift (7ndash10 nm) and a decreasein intensity (hypochromism) are observed for the bandat sim260 nm of DNA which might indicate that both thesecompounds interact with DNA [33] The shifts observedin absorbance and wavelength of DNA characteristic band


Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10

minus4 mol dmminus3 MecopropDNA +10 times 10

minus4 mol dmminus3 MCPA

(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10

minus4 mol dmminus324-D

DNA +10 times 10minus4 mol dmminus3 Dichlorprop

(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNA DNA +10 times 10

minus4 mol dmminus34-Chloro-2-methylphenol

DNA +10 times 10minus4 mol dmminus3

24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05


minus4 mol dmminus34-Chloro-2-methyl-6-nitrophenol

DNA +10 times 10minus4 mol dmminus3 Dichloro-6-nitrophenol

(d)

Figure 3 UV-Vis absorption spectra of 77 times 10minus5mol dmminus3 of DNA in the absence and presence of 10 times 10minus4mol dmminus3 of (a) mecopropand MCPA (b) 24-D and dichlorprop (c) 4-chloro-2-methylphenol and 24-dichlorophenol and (d) 4-chloro-2-methyl-6-nitrophenol and24-dichloro-6-nitrophenol in pH 72 phosphate buffer electrolyte

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05

24-DichlorophenolDNA

Sum of free TP and DNADNATP solution

(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05

4-Chloro-2-methyl-6-nitrophenol DNA


(b)

Figure 4 UV-Vis absorption spectra of 77 times 10minus5mol dmminus3 of DNA in the absence and presence of 10 times 10minus4mol dmminus3 of (a) 24-dichlorophenol and (b) 4-chloro-2-methyl-6-nitrophenol in pH 72 phosphate buffer electrolyte


Table 2 Thermodynamic parameters of DNA thermal denaturation obtained by UV spectroscopy in solutions with different herbicidesab

Compound 119905

119898∘C minusΔ(119867

260(85∘C)) Δ(Δ119867∘vH)kJmolminus1 Δ(Δ119878∘vH)kJ K

minus1molminus1

MCPA 634 plusmn 06 006 21 plusmn 8 006 plusmn 002

639 plusmn 04 007 0 0

Mecoprop 637 plusmn 06 003 7 plusmn 7 002 plusmn 002

637 plusmn 06 006 31 plusmn 8 009 plusmn 002

24-D 630 plusmn 03 0 14 plusmn 6 004 plusmn 001


Dichlorprop 641 plusmn 05 003 46 plusmn 7 013 plusmn 001


24-Dichlorophenol 643 plusmn 05 007 15 plusmn 6 004 plusmn 001


4-Chloro-2-methylphenol 643 plusmn 03 0 15 plusmn 5 004 plusmn 001


24-Dichloro-6-nitrophenol 635 plusmn 05 005 0 008 plusmn 001


4-Chloro-2-methyl-6-nitrophenol 679 plusmn 05 005 0 008 plusmn 001


a[DNA] = 77times 10minus5mol dmminus3 phosphate buffer solution ionic strength = 001mol dmminus3 For each herbicide or TP the top values correspond to a concentrationof 51 times 10minus5mol dmminus3 and the bottom values to 10 times 10minus4mol dmminus3bΔ(119867260 (85∘C)) = 119867260 (85

∘C)(DNA+herbicide or TP) minus 119867260 (85∘C)(DNA) Δ(Δ119867

∘

vH) = Δ119867∘vH (DNA+herbicide or TP) minus Δ119867∘

vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘

vH (DNA+herbicide or TP) minus Δ119878∘

vH (DNA)

can reflect the structural changes of DNA namely in thestacking pattern disruption of the hydrogen bonds betweencomplementary strands covalent binding to the DNA basesand groove binding or intercalation of aromatic rings ofmolecules between adjacent base pairs [33 36]

322 DNAMelting Studies The two strands of DNA are heldtogether mainly by stacking interactions hydrogen bondsand hydrophobic effects between the complementary basesWhen a DNA solution is heated the double helix separatesinto two single strands in a process known as DNA denatura-tion and the temperature at which the DNA strands are half-denatured is called the melting temperature 119879

119898[32] During

the disruption of the double helical structure the base-baseinteractions will be reduced increasing the UV absorbanceof DNA solution because many bases are in free form anddo not form hydrogen bonds with complementary bases[33] A shift in 119879

119898 to values different from native ds-DNA

is an indication that a drug-DNA interaction exists Themagnitude of the shift depends on the type of interactionThus for intercalating agents the increase observed in the119879

119898value is higher than in the case of agents interacting

through the DNAminor or major grooves [32]Thus UV-Visspectroscopy becomes a very valuable technique to determinethemelting temperatures and to study the interaction of smallmolecules with DNA

The DNA melting curves at 260 nm for DNA DNAherbicides and DNATPs solutions are shown in Figure 5As the absorbance obtained for the herbicides and TPs at260 nm depends on temperature all denaturation curveswere corrected to this effect by subtracting from the meltingcurves of DNAherbicides and DNATPs solutions the values

of absorbance corresponding to the solutions containing onlyherbicides or TPs Therefore the resulting curves (Figure 5)reflect only the change in absorbance due to temperatureincrease resulting from DNA denaturation The data foundallow concluding that all compounds under study reducethe hyperchromism along the DNA denaturation process Infact above 80∘C (temperature at which it is assumed thatthe strands of DNA have been totally separated) there is areduction in hyperchromism as a result of the presence ofherbicides and TPs under study with this effect being morenoticeable for the nitroderivatives (Figure 5) Moreover thehyperchromism observed is influenced by the herbicide orTP concentration an increase of concentration leads to adecrease of the DNA absorbance upon denaturation

The values of hyperchromism 119867260

calculated at 85∘Care presented in Table 2 The reduction of the hyperchromic-ity observed in the DNA melting curves indicates thatan interaction of the studied herbicides and their TPswith DNA occurs In general the interactions which occurbetween DNA and chlorophenoxy herbicides as well as theirchlorophenol transformation products are weak Howeverthe insertion of a nitro substituent on the aromatic ring(nitroderivatives) seems to induce stronger interactions Thecurves of molar fraction of denatured DNA versus tempera-ture (Figure 6) show that the denaturation temperature valuesare not significantly affected by herbicides and TPs except for4-chloro-2-methyl-6-nitrophenol (Table 2)

Spectroscopy was used to assess the thermal stabilityof the DNA in comparison with the DNAherbicide andDNATPs solutions From the calculated thermodynamicparameters it is possible to infer that the presence of theherbicides and TPs causes a slight increase in entropy and


200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA


24-Dichloro-6-nitrophenolDNA +10 times 10


(d)

Figure 5 Corrected absorbance at 260 nm versus temperature for 77 times 10minus5mol dmminus3 of DNA in the absence and presence of 10 times10minus4mol dmminus3 of (a) mecoprop and MCPA (b) 24-D and dichlorprop (c) 4-chloro-2-methylphenol and 24-dichlorophenol and (d) 4-chloro-2-methyl-6-nitrophenol and 24-dichloro-6-nitrophenol in pH 72 phosphate buffer electrolyte

enthalpy of DNAdenaturation (Table 2) In fact although thedenaturation temperature values are not significantly differ-ent (with the exception of 4-chloro-2-methyl-6-nitrophenol)the denaturation enthalpy values for DNAherbicides solu-tions are generally higher than the observed for DNAThese results suggest that a stabilization of the DNA doublehelix may occur in the presence of herbicides although noinfluence on the relative stability of base pairs is observedExternal binding to the major or minor DNA grooves canbe responsible for the observed disturbance on DNA con-formation [32 33] For the transformation product 4-chloro-2-methyl-6-nitrophenol a noteworthy increase in 119879

119898can be

observedThis is a clear indication of a strong interactionwiththe DNA molecule increasing its stability

The differing behaviour of 4-chloro-2-methyl-6-nitro-phenol compared to all other herbicides and chlorophenols

under study is in accordance with data found in the literatureIn fact the interactions of nitroaromatic compounds withDNA and the resulting mutagenic properties have beencharacterized for a variety of monocyclic polycyclic andheterocyclic nitroaromatic compounds [37] However onecan remark the different behaviour observed for 4-chloro-2-methyl-6-nitrophenol relative to 24-dichloro-6-nitrophenolThe observed differences can be explained by the positionand inherent substituent effect of the group found into 2-position of the aromatic ring Several structural factors cancontribute for the toxicity of chloronitrophenols namelythe presence of (1) an acid-dissociable group (hydroxylsubstituent) (2) strong-withdrawing moieties (halogen andnitrogroups) and (3) a bulky hydrophobic group (a ben-zene ring) The hydroxyl group decreases hydrophobicitybut increases reactivity and the addition of chloro- and


300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)


minus5 mol dmminus324-Dichlorophenol


4-Chloro-2-methylphenolDNA +50 times 10


Figure 6 Molar fraction of denatured DNA versus temperature for77 times 10minus5mol dmminus3 of DNA in the absence and presence of 50 times10minus5mol dmminus3 of TPs in pH 72 phosphate buffer electrolyte

nitrogroups increases both hydrophobicity and the acidicstrength (reactivity) of phenol The strength of the toxiceffect also stems from localization of the substituent in thearomatic nucleus Actually it is described that the positionof the chlorine substituents on the phenol ring is extremelyimportant for the observed toxicity [38] The existence of achlorosubstituent in the ortho position in phenol moleculeusually decreases its toxicity [38] Accordingly it can be onerationale to explain the toxicity outline observed for the twochloronitrophenols under study

4 Conclusion

The consequences of pesticide use on natural ecosystemsrepresent an important issue in the field of environmentalchemistry Recently it has been shown that the formation ofby-products in the environment can play a significant rolein defining the impact of pesticides on human health [3]Actually TPs can be more toxic than the parent moleculesand consequently they can represent a greater risk to humanhealth and environment

The molecular mechanisms related to the toxic effect ofchlorophenoxy herbicides and their main TPs have not beencompletely elucidated Thus in this study the evaluationof the interaction of four chlorophenoxy herbicides as wellas their main TPs with calf thymus DNA by UV-visibleabsorption spectroscopy has been accomplished Further-more the toxicity of the chlorophenoxy herbicides and TPswas also assessed evaluating their inhibitory activity towardsacetylcholinesterase

On the basis of the data obtained it seems that AChE isnot the main target of chlorophenoxy herbicides and their

TPs Nevertheless the results found revealed that the trans-formation products displayed a rather noticeable inhibitoryactivity when compared with the parent herbicides

The results obtained in the DNA-drug interaction studiesshowed for the large majority of the compounds tested aslight effect on the stability of DNA double helix Yet theexperimental data demonstrate that a transformation prod-uct (4-chloro-2-methyl-6-nitrophenol) interacts with DNAthrough a noncovalent mode Furthermore the results alsoindicate that strength of the interaction is related to the typeand localization of the substituents in the aromatic moiety

In the future attention should be given to the risksoriginating from TPs since they can pose a higher hazardthan their parent pesticides in respect to persistence bioac-cumulation and toxicity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial support from Fundacao para a Ciencia e Tecnolo-gia FCTMCTES Project PTDCAGRAAM1050442008National Funds PIDDAC also cofinanced by the EuropeanCommunity Fund FEDER through COMPETE ProgramaOperacional Factores de Competitividade (POFC) and CIQ-UP (PEst-CQUIUI00812013) is gratefully acknowledged

References

[1] FAO Global Agriculture Towards 2050 FAO Rome Italy 2009[2] S E Manahan Environmental Chemistry CRC Press Boca

Raton Fla USA 8th edition 2005[3] B I Escher and K Fenner ldquoRecent advances in environmental

risk assessment of transformation productsrdquo EnvironmentalScience and Technology vol 45 no 9 pp 3835ndash3847 2011

[4] M C R Alavanja J A Hoppin and F Kamel ldquoHealth effects ofchronic pesticide exposure cancer and neurotoxicityrdquo AnnualReview of Public Health vol 25 pp 155ndash197 2004

[5] M J Perry ldquoEffects of environmental and occupational pesti-cide exposure on human sperm a systematic reviewrdquo HumanReproduction Update vol 14 no 3 pp 233ndash242 2008

[6] V Andreu and Y Pico ldquoDetermination of pesticides and theirdegradation products in soil critical review and comparison ofmethodsrdquoTrACmdashTrends inAnalytical Chemistry vol 23 no 10-11 pp 772ndash789 2004

[7] J Cheung M J Rudolph F Burshteyn et al ldquoStructures ofhuman acetylcholinesterase in complex with pharmacologicallyimportant ligandsrdquo Journal of Medicinal Chemistry vol 55 no22 pp 10282ndash10286 2012

[8] F Kamel and J A Hoppin ldquoAssociation of pesticide expo-sure with neurologic dysfunction and diseaserdquo EnvironmentalHealth Perspectives vol 112 no 9 pp 950ndash958 2004

[9] J E Casida ldquoPest toxicology the primary mechanisms ofpesticide actionrdquoChemical Research in Toxicology vol 22 no 4pp 609ndash619 2009


[10] V JanakiDevi N NagaraniM YokeshBabu A K Kumaraguruand C M Ramakritinan ldquoA study of proteotoxicity and geno-toxicity induced by the pesticide and fungicide on marineinvertebrate (Donax faba)rdquo Chemosphere vol 90 no 3 pp1158ndash1166 2013

[11] J Y Fan J J Geng H Q Ren X R Wang and C HanldquoHerbicide Roundup and its main constituents cause oxidativestress and inhibit acetylcholinesterase in liver of Carassiusauratusrdquo Journal of Environmental Science and Health B vol 48no 10 pp 844ndash850 2013

[12] J B Chaires ldquoDrug-DNA interactionsrdquo Current Opinion inStructural Biology vol 8 no 3 pp 314ndash320 1998

[13] M F Simoniello E C Kleinsorge J A Scagnetti R A Grigo-lato G L Poletta and M A Carballo ldquoDNA damage in work-ers occupationally exposed to pesticide mixturesrdquo Journal ofApplied Toxicology vol 28 no 8 pp 957ndash965 2008

[14] F Ahmadi ldquoThe impacts of pesticides exposurerdquo in PesticidesM Stoytcheva Ed p 385 InTech Rijeka Croatia 2011

[15] Eurostat The Use of Plant Protection Products in the EuropeanUnion Data 1992ndash2003 Office for Official Publications of theEuropean Communities Luxembourg 2007

[16] T Kiely D Donaldson and A Grube Pesticides Industry Salesand Usagemdash2000 and 2001Market Estimates US Environmen-tal Protection Agency Washington DC USA 2004

[17] A Jankowska M Biesaga P Drzewicz M Trojanowicz andK Pyrzynska ldquoChromatographic separation of chlorophenoxyacid herbicides and their radiolytic degradation products inwater samplesrdquo Water Research vol 38 no 14-15 pp 3259ndash3264 2004

[18] ldquoChlorophenoxy herbicidesrdquo International Agency for Researchon Cancer vol 41 supplement 7 pp 156ndash160 1987

[19] S Chiron C Minero and D Vione ldquoOccurrence of 24-dichlorophenol and of 24-dichloro-6-nitrophenol in the Rhoneriver delta (Southern France)rdquo Environmental Science andTechnology vol 41 no 9 pp 3127ndash3133 2007

[20] S Chiron L Comoretto E Rinaldi V Maurino C Mineroand D Vione ldquoPesticide by-products in the Rhone delta(Southern France) The case of 4-chloro-2-methylphenol andof its nitroderivativerdquo Chemosphere vol 74 no 4 pp 599ndash6042009

[21] Z CHeng TOng and JNath ldquoIn vitro studies on the genotox-icity of 24-dichloro-6-nitrophenol ammonium (DCNPA) andits major metaboliterdquo Mutation ResearchmdashGenetic Toxicologyvol 368 no 2 pp 149ndash155 1996

[22] M E Reichmann S A Rice C A Thomas and P DotyldquoA further examination of the molecular weight and size ofdesoxypentose nucleic acidrdquo Journal of the American ChemicalSociety vol 76 no 11 pp 3047ndash3053 1954

[23] J Marmur ldquoA procedure for the isolation of deoxyribonucleicacid from micro-organismsrdquo Journal of Molecular Biology vol3 no 2 pp 208ndash218 1961

[24] D Habibi M A Zolfigol M Shiri and A Sedaghat ldquoNitrationof substituted phenols by different efficient heterogeneoussystemsrdquo South African Journal of Chemistry vol 59 pp 93ndash962006

[25] K Ramachandran P Sivakumar T Suganya and S Ren-ganathan ldquoProduction of biodiesel frommixed waste vegetableoil using an aluminium hydrogen sulphate as a heterogeneousacid catalystrdquo Bioresource Technology vol 102 no 15 pp 7289ndash7293 2011

[26] D M Pereira F Ferreres J Oliveira P Valentao P B Andradeand M Sottomayor ldquoTargeted metabolite analysis of Catha-ranthus roseus and its biological potentialrdquo Food and ChemicalToxicology vol 47 no 6 pp 1349ndash1354 2009

[27] J J Sramek E J Frackiewicz and N R Cutler ldquoReview of theacetylcholinesterase inhibitor galanthaminerdquo Expert Opinionon Investigational Drugs vol 9 no 10 pp 2393ndash2402 2000

[28] S Benfeito J Garrido M J Sottomayor F Borges and E MGarrido ldquoPesticides and cancer studies on the interaction ofphenoxy acid herbicides with DNArdquo in Handbook on Herbi-cides Biological Activity Classification and Health amp Environ-mental Implications D Kobayashi and E Watanabe Eds NovaScience New York NY USA 2013

[29] M G Lionetto R Caricato A Calisi and T SchettinoldquoAcetylcholinesterase inhibition as a relevant biomarker in envi-ronmental biomonitoring new insights and perspectivesrdquo inEcotoxicology Around the Globe J E Visser Ed Nova ScienceNew York NY USA 2011

[30] G L Ellman K D Courtney V Andres Jr and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no2 pp 88ndash95 1961

[31] L R Shugart ldquoDNA damage as a biomarker of exposurerdquoEcotoxicology vol 9 no 5 pp 329ndash340 2000

[32] V Gonzalez-Ruiz A I Olives M A Martın P Ribelles MT Ramos and J C Menendez ldquoAn overview of analyticaltechniques employed to evidence drug-DNA interactionsrdquo inApplications to the Design of Genosensors in Biomedical Engi-neering Trends Research and Technologies S Olsztynska EdInTech Rijeka Croatia 2011

[33] M Sirajuddin S Ali and A Badshah ldquoDrug-DNA interactionsand their study by UV-Visible fluorescence spectroscopies andcyclic voltametryrdquo Journal of Photochemistry and PhotobiologyB vol 124 pp 1ndash19 2013

[34] P S Kalsi Spectroscopy of Organic Compounds New Age Inter-national Publishers New Delhi India 2004

[35] D L Pavia G M Lampman G S Kriz and J R VyvyanIntroduction to Spectroscopy Cengage Learning Belmont CalifUSA 2009

[36] D Suh and J B Chaires ldquoCriteria for the mode of binding ofDNA binding agentsrdquo Bioorganic and Medicinal Chemistry vol3 no 6 pp 723ndash728 1995

[37] V Purohit and A K Basu ldquoMutagenicity of nitroaromaticcompoundsrdquo Chemical Research in Toxicology vol 13 no 8 pp673ndash692 2000

[38] H R Pouretedal andMH Keshavarz ldquoPrediction of toxicity ofnitroaromatic compounds through their molecular structuresrdquoJournal of the Iranian Chemical Society vol 8 no 1 pp 78ndash892011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y


Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


diseases [4 5] Although in general pesticide TPs showlower toxicity to biota than the parent compounds in somecases TPs are more toxic and represent a greater risk to theenvironment and health than the parent molecules [3 6]Therefore toxicological evaluation of pesticide TPs is consid-ered to be an emerging issue

Acetylcholinesterase enzyme (AChE) is very importantfor the central nerve system of humans and insects AChE isan enzyme that degrades through its hydrolytic activity theneurotransmitter acetylcholine and is critically important forthe regulation of neurotransmission at synapses in all areasof the nervous system [7] If AChE activity is blocked acetyl-choline accumulates at cholinergic receptor sites therebyexcessively stimulating the cholinergic receptors This canlead to various clinical complications including fibrillationleading ultimately to death [7] So large-scale inactivation ofAChE has lethal consequences for any organism with a ner-vous system It is well demonstrated that AChE is inhibitedby neurotoxins namely organophosphates and carbamatepesticides and many other compounds [8 9] In fact recentreports have also described that some fungicides and herbi-cides used in agrochemicals can also damage AChE [10 11]The evaluation of AchE inhibitory activity can help to deter-mine the toxicity levels of a diversity of net pesticides andtheir degradation products

Besides the enzymatic damage in the biologic systemspesticides have been shown to have the capacity of interactingwith the DNA Chemicals that interact with DNA can causedirect damage by covalent modifications such as adducts orstrand breaks or can perturb DNA and chromatin functionby noncovalent binding [12ndash14]Thus the preliminary evalu-ation of DNA damage is considered to be of high significancefor the study of the putative interactions of pesticides andtheir TPs with DNA and for the screening of their mutagenicproperties

Chlorophenoxy herbicides are used worldwide as plantgrowth regulators for agricultural and nonagricultural pur-poses [15 16] These herbicides can be easily transferred tosurface and ground waters due to their polar nature and rela-tively good solubility which increase the risks of contamina-tion and consequently the environmental damage [17] Basedon epidemiologic studies phenoxy acid herbicides have beenclassified by the International Agency for Research onCancer(IARC) as possibly carcinogenic to humans (category 2B)[18] The most commonly reported transformation interme-diates of phenoxy acid degradation are the correspondingchlorophenols and their nitroderivatives the latter beingenvironmentally more persistent than the parent molecules[19 20] In fact the key metabolite in the degradation ofphenoxy acids is the corresponding chlorophenol that resultsfrom cleavage of the ether bond in the parent compoundThe occurrence of nitrophenols as transformation productsis a consequence of a photochemical nitration process ofthe corresponding phenols [19] Due to the environmentaloccurrence of these compounds their risk assessment is veryrelevant because the nitration of chlorophenols reduces theiracute toxicity but the nitroderivatives could have moremarked long-term effects associated with their genotoxicity[20 21]

The aim of this work was to evaluate the interac-tion of four chlorophenoxy herbicides (MCPA meco-prop 24-D and dichlorprop) as well as their maintransformation products (24-dichlorophenol 4-chloro-2-methylphenol 24-dichloro-6-nitrophenol and 4-chloro-2-methyl-6-nitrophenol) with calf thymus DNA by UV-visibleabsorption spectroscopy (Figure 1) Thermodynamic param-eters of DNA thermal denaturation in solutions containingthe herbicides and their TPs have been evaluated to interpretthe mode of interaction Furthermore the toxicity of thechlorophenoxy herbicides and TPs was also assessed evalu-ating their inhibitory activity towards acetylcholinesterase

2 Experimental

21 Chemicals and Reagents The phenoxy acid herbicidesand the transformation products (except 4-chloro-2-methyl-6-nitrophenol that was obtained by synthesis) as well asNaCl MgCl

2sdot6H2O Na

2HPO4and NaH

2PO4were supplied

by Sigma-Aldrich (Sintra Portugal) and used without furtherpurification Bovine serum albumin (BSA) 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) acetylthiocholine iodide (ATCI)galantamine AChE (CAS 9000-81-1 EC 232-559-3) fromelectric eel (typeVI-s lyophilized powder) Tris-HCl and calfthymus DNA as Type I calf thymus DNA sodium salt (pro-tein content lt 3) were also purchased from Sigma-AldrichUltrapure (Type 1) water (Millipore Milli Q Gradient) wasused throughout the experiments

The following buffers were used in the acetyl-cholinesterase activity assay buffer A 50mM Tris-HClpH 8 buffer B 50mM Tris-HCl pH 8 containing 01BSA and buffer C 50mM Tris-HCl pH 8 containing01M NaCl and 002M MgCl

2sdot6H2O Acetylcholinesterase

from Electrophorus electricus (electric eel) (type VI-slyophilized powder 425Umg 687mgprotein) was used inthe enzymatic assay The lyophilized enzyme was dissolvedin buffer A to make 1000UmL stock solution and furtherdiluted with buffer B to get 044UmL of enzyme in themicroplate well

A stock solution (10 times 10minus3mol dmminus3) of DNA wasprepared by dissolving an appropriate amount of DNAin 005mol dmminus3 phosphate buffer solution (pH 72 ionicstrength adjusted to 01mol dmminus3 withNaCl) Stock solutions(20 times 10minus4mol dmminus3) of herbicides were prepared by disso-lution of a suitable quantity in ultrapure water

The UV absorbance measurements were performed inpH 72 phosphate buffer solutions with ionic strength001mol dmminus3 Working solutions of DNA (70 times10minus5mol dmminus3) herbicides (50 times 10minus5mol dmminus3 or 10times 10minus4mol dmminus3) and DNAherbicides were prepared bysimple dilution of the appropriate amounts of the stocksolutions in phosphate buffer and ultrapure water

The concentration of DNA solutions expressed in molesof base pairs was determined at 20∘C by UV spectroscopyat 260 nm using a molar absorption coefficient 120576

260=

13200molminus1 dm3 cmminus1 [22] A ratio of absorbance at 260 nmto that at 280 nm (119860

260119860280

) greater than 18 indicatedthat DNA was sufficiently pure and free from protein [23]


ClCl

O

R

OH

OCH3

MCPA R = H

Mecoprop R = CH3

(a)

O

R

OH

O

Cl

Cl

24-D R = H

Dichlorprop R = CH3

(b)

OH

R

Cl

R = CH3

R = Cl


24-Dichlorophenol

(c)

OH

R

Cl NO2

R = CH3

R = Cl



(d)




2Cl2(30mL) NaNO

2


2








3OH) 120575 = 230 (3H s CH

3) 752











elsewhere [28]










MCPA 129 plusmn 15



24-D 39 plusmn 003










200 220 240 260 280 300 320 340000

010

020

030

040

050

060

070

080Ab

sorb

ance

Wavelength (nm)

MecopropMCPA

Dichlorprop24-D



(a)

200 220 240 260 280 300 320 340000

020

040

060

080

100

120

140

160

Abso

rban

ce


(b)











Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




(d)


200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(b)




Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ClCl

O

R

OH

OCH3

MCPA R = H

Mecoprop R = CH3

(a)

O

R

OH

O

Cl

Cl

24-D R = H

Dichlorprop R = CH3

(b)

OH

R

Cl

R = CH3

R = Cl


24-Dichlorophenol

(c)

OH

R

Cl NO2

R = CH3

R = Cl



(d)




2Cl2(30mL) NaNO

2


2








3OH) 120575 = 230 (3H s CH

3) 752











elsewhere [28]










MCPA 129 plusmn 15



24-D 39 plusmn 003










200 220 240 260 280 300 320 340000

010

020

030

040

050

060

070

080Ab

sorb

ance

Wavelength (nm)

MecopropMCPA

Dichlorprop24-D



(a)

200 220 240 260 280 300 320 340000

020

040

060

080

100

120

140

160

Abso

rban

ce


(b)











Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




(d)


200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(b)




Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







elsewhere [28]










MCPA 129 plusmn 15



24-D 39 plusmn 003










200 220 240 260 280 300 320 340000

010

020

030

040

050

060

070

080Ab

sorb

ance

Wavelength (nm)

MecopropMCPA

Dichlorprop24-D



(a)

200 220 240 260 280 300 320 340000

020

040

060

080

100

120

140

160

Abso

rban

ce


(b)











Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




(d)


200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(b)




Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200 220 240 260 280 300 320 340000

010

020

030

040

050

060

070

080Ab

sorb

ance

Wavelength (nm)

MecopropMCPA

Dichlorprop24-D



(a)

200 220 240 260 280 300 320 340000

020

040

060

080

100

120

140

160

Abso

rban

ce


(b)











Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




(d)


200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(b)




Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(a)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05

DNADNA +10 times 10



(b)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




24-Dichlorophenol

(c)

Wavelength (nm)200 220 240 260 280 300 320 340

Abso

rban

ce

I05




(d)


200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(a)

200 220 240 260 280 300 320 340Wavelength (nm)

Abso

rban

ce

I05



(b)




Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Compound 119905

119898∘C minusΔ(119867


minus1molminus1


639 plusmn 04 007 0 0

















∘


vH (DNA) Δ(Δ119878∘

vH) =Δ119878∘


vH (DNA)



119898[32] During












200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(a)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




(b)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20




24-Dichlorophenol

(c)

200 300 400 500 600 700 800 900

000

005

010

015

020

025

030

t (∘C)

Aco

rr-A

20

DNA




(d)



119898can be





300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


300 400 500 600 700 800 900

000

020

040

060

080

100

120579

t (∘C)








4 Conclusion









Acknowledgments


References















































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Effects of Chlorophenoxy Herbicides and Their Main Transformation Products on DNA...

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Transcript of Effects of Chlorophenoxy Herbicides and Their Main Transformation Products on DNA...