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Porphyrin levels in excreta of rabbit as non-destructivebiomarkers of diazinon exposure

David Hernández-Morenoa,∗, Marcos Pérez-Lópeza, M. Prado Mígueza, Francisco Solera,Begona Jiménezb

a Toxicology Unit, Faculty of Veterinary Medicine, University of Extremadura, Avda de la Universidad s/n, 10003 Caceres, Spainb Department of Instrumental Analysis and Environmental Chemistry, Institute of Organic Chemistry, CSIC, Juan de la Cierva 3, 28006Madrid, Spain

a r t i c l e i n f o

Article history:

Received 22 February 2012

Received in revised form

14 June 2012

Accepted 23 June 2012

Available online 1 July 2012

Keywords:

Porphyrin

Rabbits

Diazinon

Non-destructive biomarkers

Pesticides

a b s t r a c t

In the present study the effect of a chronic exposition to the organophosphorous pesticide

diazinon on the porphyrin profile in feces of rabbit was evaluated, in order to validate the

use of such molecules as non-destructive biomarkers for monitoring exposure of mammals

to this environmental xenobiotic. Male and female (12:12) rabbits were exposed to oral single

doses of diazinon, feces being sampled at every 10 days, till the end of the experience (30

days). HPLC method was validated from the results obtained, for detection of porphyrins in

feces of mammals. Results revealed differences on the porphyrin profile between male and

female, the most relevant differences associated to the uroporphyrin levels. In conclusion,

porphyrin levels in rabbit’s excreta may be used as indicators of exposure to such chemi-

cals, thus providing a useful non-destructive method for monitoring exposure of animals

to different environmental pollutants. However, the effect of gender should be taken into

account when interpreting results.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

During the last decades, ecotoxicology has been increas-ingly concerned with the use of biomarkers to evaluate thebiological hazard of toxic chemicals in the environment. How-ever, in environmental contamination studies, research mayshift from evaluation of environmental health using sentinelspecies as bioindicators to a more specific investigation of the“health” of a population or an endangered species in a sit-uation of already ascertained environmental pollution. Thisinversion of terms inevitably leads to a demand for analyticaland sampling methods that are compatible with the protec-tion and conservation of the species to be studied. This is the

∗ Corresponding author at: Physiology Unit, Nursing School, University of Extremadura, Avda de la Universidad s/n, 10003 Caceres, Spain.Tel.: +34 927257450; fax: +34 927257451.

E-mail addresses: davidhm@unex.es (D. Hernández-Moreno), marcospl@unex.es (M. Pérez-López).

reason why it is essential to focus in the use of non-destructivebiomarkers (Fossi, 1994). For this purposes, some biologicalsamples (e.g., blood, feces, fur, or skin) are very interesting, asthey can be easily obtained with minimal stress to individualsor populations (Fossi et al., 1997, 1999), allowing a preliminaryrisk assessment in endangered species of wildlife.

In this sense, porphyrins are tetrapyrrolic pigments whichoccur as pigmented deposits in animal tissues and whichare ubiquitous in biological systems. They are the interme-diate metabolites of heme biosynthesis or their oxidativeby-products (coproporphyrins, uroporphyrins). They are pro-duced and accumulated in trace amounts in erythropoietictissues (i.e., relating to the formation of red blood cells), the

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liver and the kidney, and are excreted via urine or feces(Koenig et al., 2009; Lim, 1991), depending on the specific por-phyrin. Specifically, coproporphyrin and protoporphyrin areeliminated almost completely in the bile, so the study of thefecal excretion of porphyrins can be used as a measure of theamount of porphyrins made in the body (De Matteis, 1994).

It is well known that heme biosynthesis may be altered by abroad spectrum of environmental contaminants such as PCBs,dioxins, organochlorine and organophosphorous pesticides,PAHs, diphenyl ether herbicides, and heavy metals (e.g., mer-cury, arsenic, lead) (Casini et al., 2002; Jacobs et al., 1992; Marks,1985; Pain, 1989; Taira and San Martin de Viale, 1980). Thosechemical agents can thus interfere with the heme biosyn-thesis by increasing the rate of oxidation of intermediateporphyrinogens or by direct interference with enzyme activi-ties of the heme biosynthetic pathway, causing modificationsin the profile of porphyrin accumulation and excretion (Casiniet al., 2003; dos Santos et al., 2007; Kennedy and Fox, 1990;Lamola et al., 1975; Marks, 1985; Marks et al., 1987; Ng et al.,2005; Woods, 1996). Porphyrins may subsequently be iden-tified and quantified in fecal samples by high-performanceliquid chromatography (HPLC) with fluorescence detection(Lim, 1991). A change in the profile of porphyrin excretion,therefore, may serve as a useful biomarker indicating possiblealterations of the heme metabolism as a result of environ-mental contamination (Taylor et al., 2000a), confirming a closelink between the contaminant and induction of specific por-phyrins in target organs and/or tissues (Carrasco-Letelier et al.,2006; Casini et al., 2001). From a non-destructive point ofview, blood and excreta have been the most recommendedbiological materials (De Matteis, 1994) for the conservativeapproach with porphyrins, which can be of special interestfor biomonitoring of wildlife in the assessment of threatenedor endangered species (Leonzio et al., 1996). Analyses of fecalexcreta show useful information about possible toxic com-pounds that can represent a hazard for wildlife after ingestionof soil or contaminated food (Mateo et al., 2006). For example,porphyrins in birds excreta were first measured by Mirandaet al. (1986) in Japanese quail (Coturnix japonica) exposed toPCBs, and similar approaches were applied by Akins et al.(1993) with European starling (Sturnus vulgaris) and Fossi et al.(1999) or Martinez-Haro et al. (2011) with waterbirds. Withrespect to mammals, Casini et al. (2002) evaluated the healthstatus of a sea lion population using porphyrins as non-destructive biomarkers of exposure to contaminants such aspetroleum derivatives. Between all the considered samples(feces, blood, and fur), excreted porphyrins showed to be themost adequate to show the effect of contamination in sealions. In 1996, Blajeski et al. (1996) also determined the effect ofoil spill on porphyrin profile in sea otter, observing that totalporphyrins in whole fecal extracts were elevated in animalsfrom oiled sites when compared to non-oiled sites.

With these considerations, porphyrin levels in excreta maytherefore be postulated as indicators of exposure to differ-ent toxic chemicals. However, no widely distributed terrestrialspecies have been characterized as suitable models in whichto assess porphyrin profiles in the evaluation of environmen-tal contaminant exposure. In the present study, the effectof a chronic exposition to the organophosphorous pesticidediazinon on the porphyrin profile in feces of rabbit has been

evaluated, in order to validate their use as non-destructivebiomarkers for monitoring mammal exposure to this environ-mental xenobiotic.

2. Materials and methods

2.1. Reagents

Porphyrins were obtained from Frontier Scientific Europe (Lan-cashire, UK). HCl (37%) and acetonitrile (HPLC grade) were fromScharlab. The pesticide diazinon (98.3%) was obtained fromFluka. All other reagents were of analytical grade and werepurchased from Merck.

2.2. Experimental design

Twenty-four white rabbits (12/12 females/males) were individ-ually caged and acclimatized (temperature, 21 ◦C; photope-riod, 7 a.m. to 19 p.m.; food ad libitum) for 15 days beforestarting the treatment. They were then divided into threegroups for each sex (one control and two exposed) with 4animals each one. The pesticide diazinon was dissolved indistilled water prior to oral administration of both doses, cor-responding to 25 and 125 �g/kg bw (1/10 and 1/2 LD50 ofdiazinon) (Yehia et al., 2007), as a single dose at the beginningof the experience. Animals were kept during 30 days under thesame conditions that in acclimation period, and feces weresampled at the beginning, and every 10 days till the end of theexperience (30 days). All animal experiments were conductedin accordance with ethical guidelines of the European UnionCouncil (Council Directive 86/609/EEC of 24 November) and theethical committee from University of Extremadura (Spain).

2.3. Preparation of porphyrin standard solutions

The solution of porphyrins containing the type I isomersof octa-carboxylic porphyrins (uroporphyrin), hepta-, hexa-,penta-, tetra-carboxylic porphyrins (coproporphyrin), and di-carboxylic porphyrins (mesoporphyrin) carboxylic porphyrinswere prepared by dissolving an appropriate amount of por-phyrins in hydrochloric acid (3 N). Mesoporphyrin IX isincluded as a representative dicarboxylic porphyrin. Protopor-phyrin IX and coproporphyrin III were also used as standards,but they were not properly detected, thus they were excludedfrom the experiment. Dilutions of these stock solutions wereprepared with the same mixture used in the feces samples.When stored in the refrigerator, stock solutions remainedstable for at least 3 months (Kennedy et al., 1986). Severalconcentrations of a standard porphyrin solution (20.80, 41.63,83.25, 166.5, and 333 nM) were loaded into the HPLC system.

2.4. Sample preparation

Porphyrin analysis in excreta samples was performed using amodification of the analytical method reported by Mateo et al.(2004). Thus, 0.5 g of rabbit excreta (w/w) were vortexed in anEppendorf for 15 s with 0.25 ml of HCl 3 N and 0.3 ml of ace-tonitrile, and 0.3 ml of water were added in those sampleswith lowest moisture. Samples were centrifuged for 10 min

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at 16,060 RCF in an AvantiTM 30 centrifuge (Beckman Coul-ter). Supernatant was filtered using 45 �m teflon filters andtransferred to glass vials for HPLC analysis.

2.5. HPLC porphyrin determination

The HPLC method described by Lim and Peters (1984) withsome modifications was used for the identification and quan-tification of porphyrins in excreta samples.

Porphyrins were analyzed using an HPLC Jasco system con-sisting of a PU-1580 pump coupled to an HG-1580-31 mixer andan FP-920 fluorimetric detector with programmable excitationand emission wavelengths. Separation was achieved using aSupelcosil LC 18 column (25 cm × 4.6 mm and 5 �m particlesize). A 100 �l loop was used to inject the samples. The initialmobile phase was ammonium acetate (1 M, pH 5.16)/acetoni-trile (87:13, v/v), and separation was obtained using a gradientwith acetonitrile increasing from 13 to 80% in 30 min, afterthat the original rate was performed during 15 min more.The initial flow rate was 1 ml/min and changed to 1.5 ml/minbetween 17 and 30 min. A calibration curve using the stan-dard porphyrin mix showed good linearity in 7 levels from20.80 to 333 nM. The repeatability presented relative standarddeviations (RSDs) below 10% for all the porphyrins, exceptmesoporphyrin IX (11%). The reproducibility was also satis-factory, with RSD values below 10%.

The fluorescence detector was set at �ex = 400 nm,�em = 622 nm. Porphyrin concentrations in each fecal samplewere calculated with a six-point calibration curve (0–333 nM).

Measurement accuracy of the method with the HPLC sys-tem was evaluated and monitored by running porphyrinstandards prior to and during sample analysis. Similarly,extraction efficiencies were evaluated by processing aliquotsof porphyrin standards and fecal samples spiked with aliquotsof porphyrin standards through the extraction procedure. Themean recovery of fecal porphyrins was in all cases greaterthan 80% for each individual porphyrin, with coefficients ofvariation below 17%, except for 5-carboxyporphyrin (21.4%).The limit of quantification, determined as 10 times the sig-nal/noise ratio (S/N), was 5 nM for both uroporphyrin I andmesoporphyrin, and 8 nM for the rest of the considered por-phyrins.

In all cases, day-to-day coefficient of variance in retentiontimes was less than 3%.

2.6. Statistical analysis

Data were expressed as mean ± standard error. The differ-ences between the data in the control and the exposed rabbitwere analyzed by means of Mann–Whitney test, in order todetermine statistical differences among pesticide concentra-tions for every group. A probability level of less than 0.05was considered significant (95% confidence interval). More-over, a Spearman test was used to determine correlationsbetween different porphyrin isomers. Statistical analysis wasperformed with SPSS 15.0.

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3. Results and discussion

The results concerning the detection of the six different por-phyrins (initial stock solution of 588 nM) within a run timeof 40 min are presented in Fig. 1A. Similarly, Fig. 1B showsthe porphyrin pattern found in a control male feces sample.As observed, two main peaks were detected in rabbit sam-ples, corresponding to uroporphyrin I and coproporphyrin I.Although some inter-individual variations in the porphyrinratios could be observed, the general pattern remained sim-ilar. In this sense, the analysis of feces from control animalshas shown both uro- and coproporphyrin to be extremely con-sistent from animal to animal.

Figs. 2 and 3 show the variation in porphyrin (uro- andcoproporphyrins, respectively) content (nM) in fecal excreta ofboth male and female animals after diazinon exposure, com-pared to control rabbits. Total porphyrin concentrations weresubstantially higher in female than in male control rabbits.Moreover, significant differences between levels of uropor-phyrin and coproporphyrin with respect to gender could beestablished, when comparing those results concerning to con-trol animals. Control males showed lower mean uroporphyrinlevels than females (111 and 312 nM, respectively) and highermean coproporphyrin levels than females (13.5 and 6.1 nM),in both cases with statistical significance, thus revealing therelevance of gender when evaluating porphyrin profiles for

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Fig. 2 – Uroporphyrin levels in feces of rabbit(mean ± standard error) (D1: 1/10 and D2: 1/2 LD50 ofdiazinon) (*p < 0.05).

ecotoxicological purposes. In this sense, O’Halloran et al.(1988a,b) reported great variations in detected porphyrinsdepending of physiological state of birds, that may be thereason of our gender related findings in mammals.

Uroporphyrin levels in animals exposed to diazinon wereclearly decreased, compared to control rabbits. Males showeda clear inhibitory effect after the pesticide exposure in bothdoses when compared to control animals, and statisticallysignificant at 20 and 30 days after exposure (p < 0.05). Thepesticide-associated inhibitory effect was specially observedwith those animals exposed to the lowest dose of pesti-cide. Similarly, when considering females, uroporphyrin levelswere significantly lower (p < 0.05) in exposed rabbits whencompared to control animals, this inhibition being especiallyrelevant with animals exposed to the lowest dose of diazinon(p < 0.01).

No significant differences between exposed and controlgroups could be established during the whole experience withrespect to coproporphyrin.

The importance of porphyrin pattern determination in theassessment of the toxic response to exposure to porphyrino-genic compounds has been highlighted. Sometimes the totalporphyrins may remain constant even when subtle changesto the porphyrin profile can be found (Kennedy et al., 1986).

Fig. 3 – Coproporphyrin levels in feces of rabbit(mean ± standard error) (D1: 1/10 and D2: 1/2 LD50 ofdiazinon).

Spectrophotometric methods have been widely used todetermine porphyrin levels in several tissues, like blood, liver,or feces. Nevertheless, the use of more sophisticated ana-lytical methods allows identifying the porphyrin pattern. Inthis sense, in the present work, it was possible to identifythe porphyrin profile in rabbit feces applying HPLC with flu-orimetric detection. In fact, when a spectrophotofluorimetricmethod was compared with another based on HPLC, theauthors reported that even when the first one showed tobe useful for qualitative and semi-quantitative determina-tions, the results obtained were higher when HPLC were used(Fossi et al., 1996). This is in accordance with Lim and Peters(1984), who concluded that the characteristic porphyrin pro-files obtained for feces in various types of porphyrias based onHPLC techniques, provides a rapid and sensitive method forthe specific diagnosis of these diseases, from a toxicologicalpoint of view. Taylor et al. (2000b) reported that the spectroflu-orometric assay was not appropriate for the determination ofthe amounts of porphyrins in fecal samples of river otters, asfluorescent components of the fecal matrix resulted in ele-vated and erroneous values for the concentrations of bothuroporphyrin I and coproporphyrin III.

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The relevance of the present study should also be associ-ated to the validation of porphyrins as biomarkers of exposureto organophosphate pesticides, showing the physiological por-phyrin profiles. Indeed, differences in response in differentspecies, tested in several laboratory studies (Casini et al.,2003; Elliott et al., 1991; Strik, 1973), suggest that sensitivityto porphyrinogenic compounds needs to be validated for eachspecies investigated.

Our results are in agreement with those reported on ratsexposed to several doses of diazinon preparations, whichcaused disturbance on porphyrin metabolism (Bleakley et al.,1979). Uroporphyrin, 7-, 6-, and 5-carboxylporphyrin andcoproporphyrin are normally excreted in both urine and stoolwhen they are in excess, being very important to analyze thequantities of heme precursors on non-destructive samples(Daniell et al., 1997). In fact, Bleakley et al. (1979) describedelevated fecal porphyrins in rats subsequent to subchroniccutaneous exposure to diazinon. Yehia et al. (2007) reportedthat red blood cells, hemoglobin, and plasma total protein canbe significantly inhibited when rabbits are exposed to diazi-non. The porphyrinogenic effect of diazinon and its impuritieswas studied in chicken embryo liver cell cultures by Nicholet al. (1982). In this study, an increased accumulation of copro-porphyrin and to a lesser extent protoporphyrin was detectedin the tissue cultures. Other authors have reported the lack ofsignificant effect of diazinon in both blood and urinary por-phyrins of rats (Cho et al., 2003).

Some metabolites from xenobiotics are able to affect froma strong way than the original compound. This was reportedby Nichol et al. (1983), showing differences between the effectsof these chemicals on fecal porphyrins in rats. These authorsdiscovered induced levels in rats exposed to isodiazinon orto a mixture of diazinon:isodiazinon. The general lack ofinduction/inhibition on our results of porphyrins could bedue to the use of pure diazinon, instead using isodiazinon.In this sense, and in agreement to our results, 7-, 6-, and 5-carboxylporphyrin could not be detected.

The high urinary excretion of uroporphyrin has beenreported as a consequence of an inhibition of hepatic uro-porphyrinogen decarboxylase (Rizzardini and Smith, 1982).Nichol et al. (1983) theorized about this, suggesting that theincrease in porphyrins could be associated to an inhibition inporphyrinogen oxidases, obtained when rats were exposed toisodiazinon.

Some toxicity tests have failed to establish adequately therelative sensitivities of the sexes to dietary diazinon in mam-malian species. Nevertheless, there are subacute feeding trialswhich clearly reveal the greater susceptibility of the femalerat to diazinon toxicity (Davies and Holub, 1980). This greatersusceptibility was also showed in our study, where a decreasein uroporphyrin levels in females was obtained since the firstsampling.

As indicated, fecal uroporphyrin levels were higher thanthose of coproporphyrin. This result is clearly opposite tothat observed in one experiment with human samples wherehigher levels of coproporphyrin than uroporphyrin in feceswere found, being higher the levels of uroporphyrin in urine(Benton et al., 2011). Mateo et al. (2006) also reported a lackof uroporphyrin in fecal samples of geese. An explanationto such difference could be associated to sampling, because

fecal samples may contain part of urine excreta, and shouldbe taken into account when interpreting the results.

Porphyrin excretion was studied in normal human sub-jects. In agreement with our results in rabbit, taking sex intoaccount, males and females showed similar amounts of fecalcoproporphyrin fraction (Pena-Payero et al., 1980).

4. Conclusion

In conclusion, uroporphyrin levels in rabbit fecal excreta maybe used as indicators of exposure to diazinon, thus provid-ing a non-destructive method useful for monitoring wildlifeexposure to different kind of chemical pollutants.

The experimental data obtained with rabbits, designed asa long-term experiment under controlled laboratory condi-tions can be considered as a useful reference for comparisonswith biomarkers response of organisms living in polluted envi-ronments. Nevertheless, gender related variations should betaken into account when environmental samples are col-lected.

Conflict of interest statement

Nothing declared.

Acknowledgments

Authors wish to thanks the “Ministerio de Educación yCiencia” from Spain, which financially supported this work(CTM2007-60041).

Financial support was also provided by the Regional Gov-ernment of Madrid (Project P-AMB-000352-0505).

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