Embed Size (px)
Citation preview
Our reference: TIV 2553 P-authorquery-v8
AUTHOR QUERY FORM
Journal: TIV
Article Number: 2553
Please e-mail or fax your responses and any corrections to:
E-mail: [email protected]
Fax: +31 2048 52799
Dear Author,
Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen annotation in the PDFfile) or compile them in a separate list.
For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.
Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in the proof. Clickon the ‘Q’ link to go to the location in the proof.
Location inarticle
Query / Remark: click on the Q link to goPlease insert your reply or correction at the corresponding line in the proof
Q1 Bodine et al. (1983) and Guerre et al. (1994) are cited in the text but not provided in the reference list.Please provide them in the reference list or delete these citations from the text.
Thank you for your assistance.
1
2
3
4
5
6
7
9
10111213
14151617181920
2 1
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Toxicology in Vitro xxx (2011) xxx–xxx
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier .com/locate / toxinvi t
Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheralblood mononuclear cells
Umberto Bernabucci ⇑, Luciana Colavecchia, Pier Paolo Danieli, Loredana Basiricò, Nicola Lacetera,Alessandro Nardone, Bruno RonchiDepartment of Animal Science, University of Tuscia, Via S.C. De Lellis, 01100 Viterbo, Italy
a r t i c l e i n f o a b s t r a c t
2223242526272829303132
Article history:Received 5 October 2010Accepted 13 January 2011Available online xxxx
Keywords:Aflatoxin B1
Fumonisin B1
Oxidative stressPBMCDairy cattle
33343536373839
0887-2333/$ - see front matter � 2011 Published bydoi:10.1016/j.tiv.2011.01.009
⇑ Corresponding author. Tel.: +39 0761357439; faxE-mail address: [email protected] (U. Bernabucci).
Please cite this article in press as: Bernabucci, Ucells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2
Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds andreleased in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity havenot been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated.This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B1 (AFB1) andfumonisin B1 (FB1), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incu-bated for 2 and 7 days at different levels of AFB1 (0, 5 and 20 lg/ml) and FB1 (0, 35 and 70 lg/ml). Reac-tive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression ofcytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured onPBMC after incubation. The highest concentration of AFB1 and all concentrations of FB1 caused an increase(p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with20 lgAFB1/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. IntracellularSH were not affected by FB1 even though a lower (p < 0.05) SH level after 2 days exposure than after7 days was observed. MDA increased (p < 0.05) in AFB1 or FB1 treated PBMC. The exposure to FB1 for7 days increased MDA (p < 0.05) only in cells treated with 70 lg/ml. Exposure of PBMC to AFB1 reducedSOD mRNA while FB1 decreased both SOD and GSHPX-1 mRNA abundance. These results demonstratethat, even though by different mechanisms, AFB1 and FB1 may induce cytotoxicity through an impairmentof the oxidative status of PBMC.
� 2011 Published by Elsevier Ltd.
40
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
1. Introduction
Mycotoxins are secondary metabolites produced by molds andreleased in food and feed. They can cause several diseases (myco-toxicoses) in animals and humans after ingestion, skin contact orinhalation (Betina, 1989; Smith et al., 1995). The host immunity,including resistance to infectious diseases, may be affected bymycotoxins (Pestka and Bondy, 1990). Among them, aflatoxin B1
(AFB1) and fumonisin B1 (FB1) are a matter of concern due to theirwidespread contamination of cereal grain commodities, corn inparticular, and their adverse effects on human and animal health.Aflatoxin B1 is produced by toxigenic fungi belonging to the genusAspergillus and has a long history of association with illness indomesticated animals and in humans (Wogan, 1999; Bondy andPestka, 2000). Among the multitude of signs and effects due toaflatoxin exposure, the decrease in humoral and cellular immunityin farm and laboratory animals has been widely described in
75
76
77
78
Elsevier Ltd.
: +39 0761357434.
., et al. Aflatoxin B1 and fumon011.01.009
literature (Pugh et al., 1984; Bondy and Pestka, 2000). FumonisinB1 is produced by toxigenic fungi belonging to the genus Fusariumand has been associated with various diseases in animals such asequine leucoencephalomalacia, immunosuppression, porcinepulmonary oedema, liver and kidney toxicity and liver cancer aswell as human oesophageal carcinoma in some African andChinese populations (Harrison et al., 1990; Kellerman et al.,1990; Gelderblom et al., 1997). The impact of the mycotoxins onthe immune system of exposed animals is a matter of concernbecause, by this way, these natural-occurring toxins may predis-pose farm animals to the infectious diseases, which could resultin economic losses for the livestock industry (Fink-Gremmels,2008) as well as in transmission of pathogens such as Salmonellaspp. and Listeria spp. to humans (Brown et al., 1981; Atroshiet al., 1998). To date, mechanisms of action through whichmycotoxins can cause cytotoxicity are not completely clarified. Apossible mechanism is the induction of oxidative stress (Towneret al., 2003) which is classically defined as an imbalance betweenpro-oxidants and anti-oxidants in favour of the former, resultingin an overall increase in cellular levels of reactive oxygen species(Valko et al., 2007). It is well known that some mycotoxins may
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
Q1
2 U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
induce the production of free radicals and/or the reduction of anti-oxidant defenses (Shen et al., 1996; Leal et al., 1999). Since duringtoxicity no oxidative damages are often observed (Gautier et al.,2001), a first question is to establish if the onset of oxidative stressper se has to be regarded as a cause or as a consequence of the ac-tion of toxicants on the cellular system (Gagliano et al., 2006). Asimilar dilemma has been put in evidence about the relationshipbetween the uncontrolled formation of reactive oxygen speciesand the development of some pathological processes (Valkoet al., 2007).
The cytotoxic effect of mycotoxins has been studied in vivo orin vitro using different cellular models, mostly liver and kidneycells. However, data from literature reveal some rather variable ef-fects of mycotoxins on different cell systems and it has beensuggested that this may be due to different sensitivity related tothe metabolic characteristic of different cell types (Müller et al.,2004). Several in vitro trials (Bodine et al., 1984; Neldon-Ortizand Qureshi, 1992; Charoenpornsook et al., 1998) have shown thatanimal immune cells may be adversely affected by the exposure toAFB1 and FB1, even though the mechanisms through which thesemycotoxins can act have not been clearly understood yet.
Taking into account that cellular components of immune systemmay be a key target of the mycotoxins and that lymphocytes pos-sess mixed-function oxidases for transforming xenobiotics (Selkirket al., 1975), the aim of this work was to verify whether the myco-toxins aflatoxin B1 and fumonisin B1 affect the oxidative status ofbovine peripheral blood mononuclear cells (PBMC).
164
165
166
167
168
169
170
171
172
173
174
2. Materials and methods
2.1. Animals
Six healthy, not pregnant and not lactating Holstein cows of thesame parity and similar body weight (621 ± 24 kg) were used asblood donors. Blood samples were collected via jugular venipunc-ture, using 10 ml evacuated glass tubes containing sodium heparin(170 I.U.) (BD Vacutainer
�–Becton, Dickinson and Company, Ply-
muth, UK). After collection, the blood samples were stored at4 �C and promptly transferred to the laboratory.
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
2.2. Isolation and treatment of peripheral blood mononuclear cells(PBMC)
PBMC were isolated from the whole blood as previously de-scribed by Lacetera et al. (2002). After isolation, PBMC were resus-pended at a density of 1 � 106 cells/ml in RPMI-1640 enrichedculture medium containing 25 mM Hepes supplemented with10% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine,100 U/ml of penicillin G, 100 lg/ml of streptomycin sulfate and0.25 lg/ml of amphotericin B (Sigma, Milan). The time betweenthe blood collections and the establishment of cultures was lessthan 5 h.
Cells were seeded (1 ml) in duplicate at a density of 1 � 106 cell/ml in 24-well tissue culture plates under an atmosphere of 95% airand 5% CO2, and were treated with various concentration ofaflatoxin B1 (AFB1: 0.0, 5.0 and 20.0 lg/ml) or fumonisin B1 (FB1:0.0, 35.0 and 70.0 lg/ml) for 2 or 7 days at 39 �C. The concentra-tions for AFB1 were chosen on the basis of previously reports aboutthe effects of this mycotoxin on the in vitro proliferation of bovinePBMC (Bodine et al., 1983) and on adherence and phagocytic po-tential of turkey peritoneal macrophages (Neldon-Ortiz andQureshi, 1992). As far as the FB1, the level tested were selectedlooking at the values reported by Charoenpornsook et al. (1998)causing 50% inhibition of mytogen-stimulated bovine PBMC prolif-eration. These concentrations related also with in vitro studies
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
demonstrating a suppressive effect of AFB1 on inflammatory cyto-kine levels in cattle (Kurtz and Czuprynski, 1992) and cytokine pro-file alteration in pigs by FB1 (Taranu et al., 2005). The times ofexposure (2 and 7 days) were chosen taking into account the inter-est in observing the effects of the exposure to mycotoxins at twodifferent times comparable with the specific cell’s life cycle. Afterthe first 3 days of incubation, 1 ml of RPMI-1640 with 10% heatinactivated FBS was added to each well after 3 days of incubation.After 2 or 7 days of incubation, cell suspensions were transferredinto centrifuge tubes and centrifuged at 1000g for 15 min. Thesupernatants were discarded and the dry pellets (containing wholePBMC) were stored at �80 �C until the analysis.
2.3. Analysis of cell viability
Cell viability after 2 or 7 days of exposure, was determinedusing XTT assay with Cell Proliferation kit II (XTT: sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate; Roche Applied Science, Indianapo-lis, IL, USA) according to the manufacturer’s instructions. Briefly,in separate experiments, cells were seeded into 96-well micro-plates at an optimal density (106/ml) and were incubated in thesame conditions described above. For each treatment, after 2 and7 days of exposure, the cell culture medium was changed to100 ll of medium provided by the kit used, with 0.5% BSA and then50 ll of XTT labeling mixture was added to each well. After 24 hincubation at 37 �C, absorbance was measured at 450 nm. Back-ground absorbance was subtracted from each value. Results wereexpressed as optical density (OD450).
2.4. Laboratory analyses
Intracellular reactive oxygen metabolites (ROM), thiol groups(SH), malondialdehyde (MDA) and mRNA bovine cytoplasmicsuperoxide dismutase (SOD, EC 1.15.1.1) and bovine glutathioneperoxidase (GSHPX-1, EC 1.11.1.9) were assayed in PBMC culturedunder the conditions described above. Cells were lysed adding 1 mlper well of PBS solution containing Triton X-100 (0.5%) (SigmaChemical Co., St. Louis, MO, USA) and phenylmethanesulphonylfluoride (1 mM) (Sigma Chemical Co., St. Louis, MO, USA) for15 min at 4 �C. On the cell lysate, intracellular concentrations ofROM, SH and MDA were determined.
2.5. Intracellular ROM assay
The concentration of ROM was measured using a commercialkit (d-ROM test, Diacron, Grosseto, Italy) following the manufac-turer’s instructions. The kit procedure allows to determine mainlythe hydroperoxides (ROOH) contained in a biological sample. Aftera Fenton reaction, these compounds are revealed photometricallyat 505 nm and quantified using hydrogen peroxide as standard.Values were expressed as mg H2O2/dl.
2.6. Intracellular SH assay
Intracellular thiol groups were determined as described byBernabucci et al. (2002) and were expressed in lmol/l. Briefly, pro-teins of lymphocyte lysates were precipitated by adding 1 ml ofmetaphosphoric acid solution (1.67 g of metaphosphoric acid,0.2 g EDTA-disodium salt, 30 g of NaCl in 100 ml of water). Thesupernatant was separated from precipitated proteins after centri-fugation and filtration (Puradisk 25AS 0.2 lm, Whatman plc, Maid-stone Kent, UK). An aliquot of 0.5 ml of supernatant was combinedwith 0.5 ml of Na2HPO4 (300 mM). Then DTNB (5,5-dithiobis-2-nitrobenzoic acid) was added. DTNB reacts with thiols in the sam-ple to form a colored product. Sample absorbance was read at
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx 3
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
405 nm against air and the SH concentration was calculated usinga standard curve ranging from 0 to 500 lmol/l (R2 = 0.9897).
2.7. Intracellular MDA assay
Intracellular MDA concentrations were assessed by RP-HPLCafter derivatization with dinitrophenylhydrazine (DNPH) (SigmaChemical Co., St. Louis, MO, USA) at low temperature (25 �C)(Fenaille et al., 2001). Reverse phase HPLC analysis was performedusing a SpectraSystem (Thermo Separation Product, Riviera Beach,FL, USA), equipped with an Ultrasphere ODS, 250 � 4.6 mm I.D.,5 lm column (Beckman Instruments, Inc. Fullerton, CA, USA). Sam-ples and standards were injected (100 ll) and eluted isocratically(1 ml/min) at room temperature using acetonitrile/ammoniumacetate 50 mM (45:55) as mobile phase. Under this condition, thecomplex MDA-DNPH eluted at 8.2 min and peak of absorbancewas scanned at 307 nm using an UV6000 Diode Array Detector(Thermo Separation Product, Riviera Beach, FL, USA). A standardcurve was obtained following the method reported by Fenailleet al. (2001) to quantify MDA in the linear range from 0.0 to3.0 nmol/ml (R2 = 0.9989). Area peak integration and data analysiswere performed using the ChromQuest™ 3.0 software package(ThermoQuest Inc, San Jose, CA, USA). The detection limit(LOD = 0.0008 nmol/ml) of the HPLC method was calculated usinga signal-to-noise ratio of 3:1 for a standard solution; similarly thequantitation limit (LOQ = 0.0026 nmol/ml) was estimated on a sig-nal-to-noise ratio of 10:1.
2.8. SOD and GSHPX-1 mRNA abundance
Total RNA was isolated by homogenizing lymphocyte pellet in1 ml of TRI-REAGENT™ following the procedure described by themanufacturer (Sigma–Aldrich, Milano, Italy). Quantification ofSOD and GSHPX-1 mRNA were carried out by ribonuclease protec-tion assay (RPA) as previously described by Bernabucci et al.(2004). After extraction, total RNA was quantified by measuringits absorbance at 260 nm and stored at �80 �C until the RPA. Spe-cific antisense ribonucleotide probes were generated using cDNAsof SOD, GSHPX-1 and glyceraldehyde-3-phosphate dehydrogenase(GAPDH), which was used as internal control. cDNAs were pro-duced from bovine lymphocyte RNA by reverse transcription-poly-merase chain reaction (RT-PCR). The different primers weredesigned using published SOD, GSHPX-1 and GAPDH bovine nu-cleic acid sequences. All necessary details about the primers arelisted in Table 1. Reverse transcription reaction was carried out(Superscript™ II kit, GIBCO-BRL, Life Technologies, USA) accordingto the manufactures instructions. The total PCR reaction mixture of100 ll contained 5 units of Taq DNA polymerase (GIBCO-BRL, LifeTechnologies, USA). To check for specificity, PCR products wereanalyzed by gel electrophoresis. The PCR products, containing se-quence of the T7 promoter at the 50 end, were transcribedin vitro directly using a Maxiscript transcription Kit (Ambion,Inc., Austin, TX, USA) according to the manufacturer’s instructions.After transcription, all riboprobes were purified and labeled with
Table 1Sequences of PCR primers, position in coding sequence (CDS), PCR product length and Gesequence of T7 promoter for reverse primers only was: TAATACGACTCACTATAGGGAG.
Gene Primer sequence (50?30)
SOD Forward TGGAGACAATACACAAGGCTGReverse CTGCCCAAGTCATCTGGTTT
GSHPX-1 Forward AACGCCAAGAACGAGGAGATReverse GGACAGCAGGGTTTCAATGT
GAPDH Forward TCATCCCTGCTTCTACTGGCReverse CCTGCTTCACCACCTTCTTG
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
biotin using Brighstar™ Psoralen-Biotin Kit (Ambion, Inc., Austin,TX, USA) according to the manufacturer’s instructions. Multiple-probe RPAs were carried out using the RPA III™ kit (Ambion, Inc.,Austin, TX, USA) as described for the standard procedure. TargetRNA samples and riboprobes were co-precipitated with ammo-nium acetate and ethanol. Yeast RNA from the RPA III™ kit wasused as negative control. The RNA samples and riboprobes weresubsequently processed following the procedure described by themanufacturer (Ambion, Inc., Austin, TX, USA). For the quantitativeanalysis of SOD and GSHPX-1 mRNAs, known amounts of in vitrosynthesized SOD and GSHPX-1 sense RNA were hybridized withan excess of labeled antisense probes to construct the standardcurves. The samples were loaded on acrylamide gel and then elec-trophoretically transferred to a positively-charged nylon mem-brane (BrightStar™-Plus, Ambion, Inc., Austin, TX, USA). ThemRNA was cross-linked to the wet membrane after the transfer.The nonisotopic detection of the probe fragments protected wasperformed using BrightStar™ and BioDetect™ kits (Ambion, Inc.,Austin, TX, USA) following the procedure described by the manu-facturer. Chemiluminescent films were analyzed with the KodakEDAS-290 densitometer and ID Image Analysis software (EastmanKodak Company, Rochester, NY, USA). Samples were analyzed inconjunction with the standard curve and the intensity of the probefragments protected by unknown samples was compared to thestandard curve to determine the absolute amounts (pg/10 lg TotalRNA) of SOD and GSHPX-1, mRNA.
2.9. Statistical analysis
Data for all variables measured were analyzed as repeated mea-sures using GLM procedure of SAS
�(SAS, 1999). Statistical analysis
was done separately for each mycotoxin tested. The model in-cluded fixed effects: mean of random cow effect, mean effect ofmycotoxin concentrations (0, 5 and 20 for AFB1 and 0, 35 and 70for FB1), mean effect of time of exposure (2 or 7 days), interactionmycotoxin concentration � time, and the error term or unex-plained residual element. Least square means were separated withthe predicted difference (PDIFF) procedure. Data are reported asleast-square means with standard errors. Significance was declaredat p < 0.05.
3. Results
3.1. Cell viability
PBMC viability was evaluated in the presence of AFB1 and FB1
using the XTT test. Both mytoxins did not diminish cell viabilityin a concentration or time-dependent manner (Table 2). Cell viabil-ity was independent by the FB1 concentration (0, 35 and 70 lg/ml)while, at the highest concentration tested (20 lg/ml), AFB1 ex-posed cells were more viable compared with PBMC exposed to 0and 5 lg/ml. For both mycotoxins, cell viability was higher in cellsincubated for seven compared with cells incubated for 2 days.
neBank accession number of the used published bovine nucleic acid sequences. The
CDS Length GeneBank
147 234 X54799380265 336 X13684600581 177 U85042757
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
Table 2Cell viability of lymphocytes exposed for 2 or 7 days to different concentration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1). Data are expressed as Lsmean ± SEM of the opticaldensities recorded at 450 nm.
AFB1 FB1
0 lg/ml 5 lg/ml 20 lg/ml 0 lg/ml 35 lg/ml 70 lg/ml
Time2 days 0.523 ± 0.006b 0.545 ± 0.001b 0.594 ± 0.005a 0.535 ± 0.007 0.562 ± 0.006 0.788 ± 0.0067 days 0.775 ± 0.029b 0.767 ± 0.029b 0.831 ± 0.022a 0.760 ± 0.014 0.743 ± 0.006 0.796 ± 0.007Time effect ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄
a,b Significant differences (p < 0.05) between concentrations within the day of exposure.* Significant differences (p < 0.05) between days of exposure within toxin concentration.*** Significant differences (p < 0.001) between days of exposure within toxin concentration.
4 U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
3.2. Intracellular ROM
To assess the effect of exposure to AFB1 or FB1 on the genesis ofhighly oxidant metabolites in PBMC, intracellular ROM was deter-mined after incubation with mycotoxins. Compared with the con-trol, an increase (p < 0.05) of intracellular concentration of ROMwas observed at the highest concentration of AFB1 and at both con-centrations of FB1 tested without any time-dependent effect(Fig. 1A and B).
313
314
315
316
3.3. Intracellular SH
To evaluate the intracellular depletive potential of both myco-toxins, intracellular thiols were measured on PBMC after incuba-
0.40
0.45
0.50
0.55
0.60
0 5 20
AFB1 concentration (µg/ml)
aa a
a
bb
RO
M c
onc.
(m
g H
2O2/d
l)
A
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
0 35 70FB1 concentration (µg/ml)
2 days7 days
a
b b
a
bb
RO
M c
onc.
(m
g H
2O2/d
l)
B
2 days7 days
Fig. 1. Intracellular reactive oxygen metabolites (ROM) concentrations in PBMCexposed for 2 or 7 days to different concentration of (A) aflatoxin B1 (AFB1) and (B)fumonisin B1 (FB1). Data are expressed as Lsmean ± SEM. a,bSignificant differences(p < 0.05) between concentrations within the day of exposure.
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
tion either with AFB1 or FB1. Intracellular SH decreased at20 lgAFB1/ml (p < 0.05) compared to the control, with a significantmore strong effect observed after 7 days of exposure (Fig. 2A). Noeffect of FB1 concentration on intracellular SH was observed(Fig. 2B). Compared with the 7 days exposure, intracellular thiolswere slightly lowered (p < 0.05) after 2 days of exposure to FB1,regardless the concentration tested (Fig. 2B).
3.4. Intracellular MDA
To determine whether the exposure of PBMC to AFB1 or FB1 canenhance the lipoperoxidative activity of PBMC, malondialdehyde,an end-product of peroxidative pathways of lipids, was selectively
60
80
100
120
140
160
180
200
0 5 20AFB1 concentration (µg/ml)
SH c
onc.
(µm
ol/l)
2 days7 days
b bb
b
a
a
*
A
60
80
100
120
140
160
180
200
0 35 70
FB1 concentration (µg/ml)
2 days7 days
*
*
SH c
onc.
(µm
ol/l)
B
Fig. 2. Intracellular thiols (SH) level in PBMC exposed for 2 or 7 days to differentconcentration of (A) aflatoxin B1 (AFB1) and (B) fumonisin B1 (FB1). Data areexpressed as Lsmean ± SEM. a,bSignificant differences (p < 0.05) between concen-trations within the day of exposure. ⁄Significant differences (p < 0.05) between daysof exposure within toxin concentration.
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
ab
a
*
A
20
25
30
35
40
45
SOD
mR
NA
b b
2 days7 days
U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx 5
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
quantified by RP-HPLC. Intracellular MDA increased (p < 0.05) in adose dependent manner in cells treated with AFB1 both after 2 and7 days of exposure (Fig. 3A). After 2 days of exposure, FB1 causedan increase (p < 0.05) of MDA in a dose dependent manner(Fig. 3B). After 7 days of treatment, the MDA level increased(p < 0.05) only in cells exposed to the highest FB1 concentrationtested (Fig. 3B).
a b
a
AFB1 concentration (µg/ml)
0
5
10
15
0 5 20
a
10
15
20
25
SOD
mR
NA
b
***
30 2 days7 days
B
3.5. SOD and GSHPX-1 mRNA
Effects of AFB1 and FB1 on PBMC SOD mRNA abundance arereported in Fig. 4. Exposure of PMBC to the highest level ofAFB1 led to a reduction (p < 0.05) of SOD mRNA abundance bothafter 2 and 7 days of exposure even though at the longest timeof exposure, a significant decrease was recorded already at5 lgAFB1/ml (Fig. 4A). A significant dose-dependent reductionof SOD mRNA abundance (p < 0.05) was observed exposing PBMCto FB1 for 7 days regardless the level tested (Fig. 4B). Geneexpression of GSHPX-1 was moderately decreased by exposureto 5 lgAFB1/ml for 2 days (Fig. 5A) whereas a strong effect wasrecorded in cells exposed for 2 or 7 days to FB1 (Fig. 5B), withthe lowest values of expression recorded in cells incubated for7 days.
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0 5 20
AFB1 concentration (µ g/ml)
MD
A c
onc.
(nm
ol/m
l)
2 days7 days
*
b
c
b
a a
c
A
0.00
0.01
0.02
0.03
0.04
0 35 70
FB1 concentration (µg/ml)
MD
A c
onc.
(nm
ol/m
l)
aa
b
a
c
b*
*
B 2 days7 days
Fig. 3. Intracellular malondialdehyde (MDA) concentrations in lymphocytesexposed for 2 or 7 days to different concentration of (A) aflatoxin B1 (AFB1) and(B) fumonisin B1(FB1). Data are expressed as Lsmean ± SEM. a,b,cSignificant differ-ences (p < 0.05) between concentrations within the day of exposure. ⁄Significantdifferences (p < 0.05) between days of exposure within toxin concentration.
0
5
0 35 70
FB1 concentration (µ g/ml)
aa
Fig. 4. Messenger RNA abundance of cytoplasmic superoxide dismutase (SOD) fromPBMC exposed for 2 or 7 days to a different concentration of (A) aflatoxin B1 (AFB1)and (B) fumonisin B1 (FB1). mRNA is expressed as pg/10 lg total RNA. Data areexpressed as Lsmean ± SEM. a,bSignificant differences (p < 0.05) between concen-trations within the day of exposure. ⁄Significant differences (p < 0.05) between daysof exposure within toxin concentration. ⁄⁄Significant differences (p < 0.01) betweendays of exposure within toxin concentration.
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
4. Discussion
Viability of AFB1 and FB1 treated cells, was not reduced com-pared with the untreated controls. On the contrary, increased via-bility was observed in cells exposed to the highest AFB1
concentration (20 lg/ml). Moreover, cell viability was higher after7 days than after 2 days of exposure for all AFB1 or FB1 concentra-tions tested, indicating that a moderate but significant cell prolifer-ation occurred in our experimental conditions. Results obtained oncell viability authorize us to exclude cell death as a co-factor ininducing changes of the parameters measured.
The ROM content in animal cells may be increased by severalfactors, including metabolism of xenobiotics, such as the mycotox-ins, with the onset of oxidative stress conditions as a result (Klau-nig and Kamendulis, 2001). Several authors (Abado-Becognee et al.,1998; Yin et al., 1998; Abel and Gelderblom, 1998; Leal et al., 1999)reported that some mycotoxins can cause cell membrane damagethrough the increase of lipid peroxidation. Moreover, results ofthose studies indicate the important role of the ROM productionin explaining the cytotoxic and, possibly, genotoxic potential ofthe mycotoxins. At exposure levels of AFB1 comparable to thosetested in the present study, Bodine et al. (1984) reported a strongreduction of the blastogenic potential in bovine lymphocytes (up to90% respect to the control), hypothesizing a general inhibition of Tlymphocyte functions, such as killer, helper, effector or other im-mune processes which may compromise the immunological sur-veillance mechanisms. Our data show a dose-dependent increaseof ROM in bovine PBMC, especially under FB1 exposure conditions.Looking at ROM as determinants of the oxidative stress in PBMC, it
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
a a
b
0
2
4
6
8
10
12
0 35 70
GSH
PX
-1m
RN
A
FB1 concentration (µ g/ml)
b
a
b
aa
a
*
5
10
15
20
AFB1 concentration (µg/ml)
GSH
PX
-1m
RN
A
A
*
2 days7 days
B
*
0
25
0 5 20
a
bb
2 days7 days
Fig. 5. Messenger RNA abundance of glutathione peroxidase (GSHPX-1) from PBMCexposed for 2 or 7 days to a different concentration of (A) aflatoxin B1 (AFB1) and (B)fumonisin B1(FB1). mRNA is expressed as pg/10 lg total RNA. Data are expressed asLsmean ± SEM. a,bSignificant differences (p < 0.05) between concentrations withinthe day of exposure. ⁄Significant differences (p < 0.05) between days of exposurewithin toxin concentration.
6 U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
is useful to take into account that their increase may be the expres-sion of a true overproduction or a deficiency of cellular enzymaticand non-enzymatic anti-oxidants (Valko et al., 2007). Among ROM,the superoxide anion radical ðO��2 Þ has to be considered as a ‘‘pri-mary’’ reactive oxygen species (Valko et al., 2006). The main siteof the ðO��2 Þ production lies in the mitochondria (Cadenas and Sies,1998) due to a ‘‘leakage’’ of the electron transport chain (Valkoet al., 2007). C2-ceramide (N-acetylsphingosine) was previously re-ported to affect the mitochondrial electron transport chain (García-Ruiz et al., 1997), making possible an overproduction of hydrogenperoxide (H2O2) and ROM. Bionda et al. (2004) studying rat livermitochondria, demonstrated that the inner as well as the outermitochondrial membranes are sites of ceramide synthase activityin vitro and also that such activity could be modulated by FB1 atconcentrations ranging from 10 up to 50 lM (i.e. from 7.2 to36.1 lgFB1/ml). Interestingly, those researchers showed that theexposure to FB1 (generally recognized as an inhibiting agent ofthe ceramide synthesis) led to a dose-dependent increase of themitochondrial C2-ceramide synthesis. Even though obtainedthrough different experimental models, taken together these find-ings authorize the hypothesis that, under FB1 exposure, a truemitochondrial over production and release of superoxide ion orother ROM may occur. In supporting such hypothesis, Kouadioet al. (2005) showed that FB1 affects in vitro the functionality ofCaco-2 cell mitochondria inducing lipid peroxidation and MDAproduction.
The inactivation of free radicals is the main mechanism to con-trast damages due to oxidation (Atroshi et al., 1997; Leal et al.,1999). Among other cellular scavengers, glutathione (GSH) plays
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
a primary role and it should be regarded as an early biological mar-ker of the oxidative stress (Gagliano et al., 2006). Since the totalGSH represent more than 95% of all non-protein thiolic groups ofthe cell (van den Berg et al., 1992; Lakritz et al., 2002), the measureof the cell’s SH content provides a good estimation of the GSH it-self. In our study, the intracellular thiols showed a dose-dependentreduction after incubating the PBMC with AFB1 but not with FB1. Aswell known, metabolism of AFB1 includes the epoxidation of the8,9-double bond and the hydroxylation of both furan and lactonerings together with oxidative demethylation, resulting in the for-mation of a variety of polar metabolites. via the action of glutathi-one-S-transferase, these metabolites are mainly conjugated withGSH before to be excreted (Degan and Neuman, 1978). Therefore,as a consequence of the cell exposure to AFB1, a high amount ofGSH is needed for the catalysed conjugation reactions. In fact,inhibiting the GSH synthesis in vivo, Hayes et al. (1998) sensitisedrats to the genotoxic effects of AFB1 due to the depletion ofglutathione.
In the present study, we did not find any significant effect onintracellular SH treating PBMC with FB1 for 2 or 7 days. The expo-sure to FB1 has been reported to exert variable and sometimes notcomparable effects on GSH levels in different cell types (Kang andAlexander, 1996; Lim et al., 1996; Atroshi et al., 1999; Šegvic-Klaricet al., 2006). The lack of univocal effects on the cell’s thiol contentafter exposure to FB1, could be partly due to the different ability ofspecific cell types to maintain a suitable level of intracellular GSHand eventually to manage the oxidative stress. In fact, the mainte-nance of cellular GSH level and cellular redox state is a dynamicprocess achieved by the cell balancing the rate of GSH synthesis/regeneration (Han et al., 2000) on one side and the GSH utilizationor GSH/GSSG efflux (Coppola and Ghibelli, 2000) on the other. Inparticular, the de novo GSH synthesis is regulated trough a rate lim-iting catalysis involving the c-glutamylcysteine synthetase (c-GCS)(Morales et al., 1997) whose expression was found to differ widelyamong different cell types (Rumora et al., 2007). On the other hand,fumonisins do not undergo any sort of significant metabolizationin mammal cells producing polar metabolites (Abel and Gelderb-lom, 1998) and needing GSH for the conjugation reactions. Lookingat the complexity of such scenario, it is not surprisingly that the ef-fect of FB1 on the cell GSH content reported in literature is quitevariable depending upon the type of cell studied (Stockmann-Juv-ala et al., 2004; Rumora et al., 2007). Our data do not permit toestablish whether mycotoxins differentially affect different celltypes represented within the population of bovine PBMC. On theother hand, our study was not aimed at assessing that. Studieson the effects of mycotoxins in ruminants PBMC are at a very earlystage and in that context our study provided original data. Further-more, our data justify further studies aimed at assessing whetherthe effects of mycotoxins on PBMC may be different in the differentcell types represented within the PBMC population.
In our experimental conditions, despite the overproduction ofROM, intracellular SH did not significantly decrease under FB1
exposure. In contrast, FB1 exposure reduced the abundance ofGSHPX-1 mRNA (up to 70%). Since GSHPX-1 is directly involvedin transforming some oxidizing agents such as hydrogen peroxideor some lipo-peroxides (LOOH) in low reactive compounds usingGSH (Valko et al., 2006), its decreased expression and consequentlyits activity, might explain the low thiols depletion observed, evenin the presence of high level of ROM. In our study, low but statis-tically significant differences of the intracellular SH content inPBMC exposed for 2 or 7 days were observed. A similar time-dependent response under the exposure to FB1 has been reportedby Stockmann-Juvala et al. (2004) in rat C6 glioblastoma cells butnot in other cell lines. In agreement with Šegvic-Klaric et al.(2006), probably due to different and sometimes not comparabletime- and cell-dependent responses, our data confirm that cell re-
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598
U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx 7
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
sponse, in term of GSH content after FB1 exposure is not conclusiveand needs further research.
The MDA production is recognized as an important factor indetermining alteration of membrane fluidity (Chen and Yu, 1994;Ferrante et al., 2002) and increase of membrane fragility accompa-nying final cell death (Halliwell and Chirico, 1993). Moreover, assome other aldehydes produced by lipid peroxidation processes,MDA inhibits various enzyme reactions and exerts a strong inhib-itory effect on the synthesis of DNA and proteins (Esterbauer et al.,1982). Trough the interaction with DNA, MDA is able to producesome adducts that are thought to play a role in the cancer process(Marnett, 1999). Wang and Liehr (1995) provided evidence for theexistence of MDA adducts to deoxyadenosine monophosphate(dAMP) as well as deoxyguanosine monophosphate (dGMP) inmammals. Vaca et al. (1995) found the same adducts in man’swhite cells and breast. Looking at the effect of mycotoxins on thecellular MDA concentration, Baudrimont et al. (1997) showed agood correlation between the measurement of MDA and the in-crease of lipid peroxidation in Vero cells exposed to OchratoxinA, whereas Stockmann-Juvala et al. (2004) described an increaseof MDA in two rodent (rat C6 glioblastoma and mouse GT1-7 hypo-thalamic) and a human (SH-SY5Y neuroblastoma) cell lines ex-posed to increasing levels of FB1. As previously stated by Abado-Becognee et al. (1998), the lipid peroxidation products can be con-sidered as a very sensitive markers of exposure to FB1. Our results,obtained exposing PBMC to AFB1 or FB1, clearly show a dose-dependent increase of the cellular MDA concentration indicatinga possible oxidative stress induction exerted by both toxins and,in the case of FB1, this seems to happen even without depletionof intracellular SH. The dramatic increase of MDA, observed afterthe exposure to AFB1, is in line with what reported in vivo by others(Guerre et al., 1994; Sivanesan and Begum, 2007). Taking together,these facts are in agreement with the theory that considers MDA asa late biomarker of oxidative stress and cellular damage, regardlessof the way they are induced (Vaca et al., 1988). The increased dose-dependent production of MDA, could explain the high cytotoxicand genotoxic potential of AFB1 and FB1 as reported by severalauthors (Abado-Becognee et al., 1998; Guerre et al., 1999; De Lore-nzi et al., 2005; Alpsoy et al., 2009).
Results of the present study showed a general decrease of geneexpression of antioxidant enzymes (SOD and GSHPX-1) followingthe treatment with AFB1 and FB1. Even though the mRNA abun-dance did not reflect the activity of SOD and GSHPX-1, the reduc-tion of their gene expression at all concentrations and times ofexposure for AFB1 and only at 7 days of exposure for FB1, mightbe considered as the result of the well known inhibitory effect ofsome mycotoxins on the synthesis of RNA and proteins (Eriksenet al., 2004; Verma, 2004). Csonka et al. (2000) reported that, underoxidative stress conditions, rat heart cells showed a decrease of theSOD mRNA and of the activities of SOD and GSH peroxidase. Eventhough the mechanisms have to be fully clarified, a sort of tran-scriptional effect due to exposure to mycotoxins seems highlyprobable. Looking at the GSHPX-1 abundance in treated PBMC, itis interesting to notice that under the AFB1 exposure a low intracel-lular level of SH was observed together with a partially reducedGSHPX-1 gene expression. Since the activity of glutathione perox-idase is thought to be GHS-dependent (Meister and Anderson,1983; Alpsoy et al., 2009), a reduced content of intracellular GSHtogether with an under-expression of the enzyme GSHPX-1, couldsynergically affect the inter-conversion rate of lipid peroxil radicals(LOOd) to LOH (Valko et al., 2006). This hypotheses could explainthe high MDA production caused by the exposure to AFB1. In fact,it is most probable that when the inter-conversion of LOOd to LOHdecreases due to the GSH depletion and/or due to the low activityof GSH peroxidase, a great part of the peroxil radical pool gener-ated under oxidative conditions may be addressed toward the syn-
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
thesis of MDA, 4-hydroxynonenal (HNE) and other end-products ofthe lipid-peroxidation (Valko et al., 2007).
In conclusion, results of the present study show that both AFB1
and FB1 have affected the oxidative status of bovine PBMC and theexpression of genes codifying for enzymes controlling ROM: SODand GSHPX-1. Among markers of the oxidative status measured,MDA concentration gave an univocal and clear estimation of thebovine PBMC response to mycotoxin exposure.
The induction of oxidative stress may be seen as a direct resultof exposure to both mycotoxins implying a reduced availability ofantioxidant defences and possibly leading to cellular damages.Looking at the cause-effects relationship, using MDA as biomarkerof oxidative stress, the study outlines that the exposure to AFB1 isable to induce stronger oxidative status imbalance in PBMC at low-er concentration than FB1, in agreement with its well known high-est cytotoxic potential.
Due to the wide presence of AFB1 and FB1 as contaminants com-monly occurring in feed and food, further studies should be neces-sarily to fully understand to what extent the alteration of theoxidative status of lymphocytes may be responsible of an impairedimmune response in animals exposed and co-exposed to thesemycotoxins.
Acknowledgments
This work was financially supported by MiPAF (DM 41775/2001) and by the University of Tuscia (RSA 2004). The authorsthank Dr. D. Scalia for the technical support.
References
Abado-Becognee, K., Mobio, T.A., Ennamany, R., Fleurat-Lessard, F., Shier, W.T.,Badria, F., Creppy, E.E., 1998. Cytotoxicity of fumonisin B1: implication of lipidperoxidation and inhibition of protein and DNA syntheses. Arch. Toxicol. 72,233–234.
Abel, S., Gelderblom, W.C.A., 1998. Oxidative damage and fumonisin B1–inducedtoxicity in primary rat hepatocytes and rat liver in vivo. Toxicology 131, 121–131.
Alpsoy, L., Yildirim, A., Agar, G., 2009. The antioxidant effects of vitamin A, C and Eon aflatoxin B1-induces oxidative stress in human lymphocytes. Toxicol. Ind.Health 25, 121–127.
Atroshi, F., Rizzo, A., Biese, I., Veijalainen, P., Saloniemi, H., Sankari, S., Andersson, K.,1999. Fumonisin B1-induced DNA damage in rat liver and spleen: effects ofpretreatment with Coenzyme Q10, L-Carnitine, a-Tocopherol and Selenium.Pharmacol. Res. 40, 459–467.
Atroshi, F., Rizzo, A., Westermarck, T., Ali-Vehmas, T., 1998. Effects of tamoxifen,melatonin, coenzyme Q10, and L-carnitine supplementation on bacterialgrowth in the presence of mycotoxins. Pharmacol. Res. 38, 289–295.
Atroshi, F., Rizzo, A., Biese, I., Veijalainen, P., Antila, E., Westermarck, T., 1997. T-2toxin-induced DNA damage in mouse livers: the effects of pretreatment withcoenzyme Q10 and a-tocopherol. Mol. Aspects Med. 18, s255–s258.
Baudrimont, I., Ahouandjivo, R., Creppy, E.E., 1997. Prevention of lipid peroxidationinduced by ochratoxin A in Vero cells in culture by several agents. Chem. Biol.Interact. 104, 29–40.
Bernabucci, U., Ronchi, B., Basificò, L., Pirazzi, D., Rueca, F., Lacetera, N., Nardone, A.,2004. Abundance of mRNA of apolipoprotein B100, apolipoprotein E andmicrosomal triglyceride transfer protein in liver from periparturient dairycows. J. Dairy Sci. 87, 2881–2888.
Bernabucci, U., Ronchi, B., Lacetera, N., Nardone, A., 2002. Markers of oxidativestatus in plasma and erythrocytes of transition dairy cows during hot season. J.Dairy Sci. 85, 2173–2179.
Betina, V., 1989. Structure-activity relationships among mycotoxins. Chem. Biol.Interact. 71, 105–146.
Bionda, C., Portoukalian, J., Schmitt, D., Rodriguez-Lafrasse, C., Ardail, D., 2004.Subcellular compartmentalization of ceramide metabolism: MAM(mitochondria-associated membrane) and/or mitochondria? Biochem. J. 382,527–533.
Bodine, A.B., Fisher, S.F., Gangjee, S., 1984. Effect of aflatoxin B1 and majormetabolites on phytohemeagglutinin-stimulated lymphoblastogenesis ofbovine lymphocytes. J. Dairy Sci. 67, 110–114.
Bondy, G.S., Pestka, J.J., 2000. Immunomodulation by fungal toxins. J. Toxicol.Environ. Health Part B, Crit. Rev. 3, 109–143.
Brown, V.M.D., Pier, A.C., Richard, J.L., Krogstad, R.E., 1981. Effects of dietaryaflatoxin on exiting bacterial intramammaru infections in dairy cows. Am. J.Vet. Res. 42, 927–933.
Cadenas, E., Sies, H., 1998. The lag phase. Free Radic. Res. 28, 601–609.
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631632633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670671672673674675676677
678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738739740741742743744745746747748749750751752753754755756
8 U. Bernabucci et al. / Toxicology in Vitro xxx (2011) xxx–xxx
TIV 2553 No. of Pages 9, Model 5G
25 January 2011
Charoenpornsook, K., Fitzpatrick, J.L., Smith, J.E., 1998. The effects of fourmycotoxins on the mitogen stimulated proliferation of bovine peripheralblood mononuclear cells in vitro. Mycopathologia 143, 105–111.
Chen, J.J., Yu, B.P., 1994. Alteration in mitochondrial membrane fluidity by lipidperoxidation products. Free Radic. Biol. Med. 17, 411–418.
Coppola, S., Ghibelli, L., 2000. GSH extrusion and the mitochondrial pathway ofapoptotic signalling. Biochem. Soc. Trans. 28, 56–61.
Csonka, C., Pataki, T., Kovacs, P., Muller, S.B., Schroeter, M.L., Tosaki, A., Blasig, I.E.,2000. Effects of oxidative stress on the expression of antioxidative defenceenzymes in the spontaneously hypertensive rat hearts. Free Rad. Biol. Med. 29,612–619.
De Lorenzi, L., De Giovanni, A., Malagutti, L., Molteni, L., Sciaraffia, F., Tamburini, A.,2005. Genotoxic activity of the Fumonisin B1 mycotoxin in cultures of bovinelymphocytes. Ital. J. Anim. Sci. 4, 395–402.
Degan, D.H., Neuman, H.G., 1978. The major metabolite of AFB1 in the rat is aglutathione conjugate. Chem. Biol. Interact. 22, 239–255.
Eriksen, G.S., Pettersson, H., Lundh, T., 2004. Comparative cytotoxicity ofdeoxynivalenol, nivalenol and their acetylated derivatives and deepoxymetabolites. Food Chem. Toxicol. 42, 619–624.
Esterbauer, H., Cheeseman, K.H., Dianzani, M.U., Poli, G., Slater, T.F., 1982.Separation and characterization of the aldehydic products of lipidperoxidation stimulated by ADP-Fe2+ in rat liver microsomes. Biochem. J. 208,129–140.
Fenaille, F., Mottier, P., Turesky, R.J., Ali, S., Guy, P.A., 2001. Comparison of analyticaltechniques to quantify malondialdehyde in milk powder. J. Chromatogr. A 921,237–245.
Ferrante, M.C., Meli, R., Mattace Raso, G., Esposito, E., Severino, L., Di Carlo, G.,Lucisano, A., 2002. Effect of fumonisin B1 on structure and function ofmacrophage plasma membrane. Toxicol. Lett. 129, 181–187.
Fink-Gremmels, J., 2008. The role of mycotoxins in the health and performance ofdairy cows. Vet. J. 176, 84–92.
Gagliano, N., Dalle Donne, I., Torri, C., Migliori, M., Grizzi, F., Milzani, A., Filippi, C.,Annoni, G., Colombo, P., Costa, F., Cava Grimaldi, G., Bertelli, A.A.E., Giovannini,L., Gioia, M., 2006. Early cytotoxic effects of Ochratoxin A in rat liver: amorphological, biochemical and molecular study. Toxicology 225, 214–224.
García-Ruiz, C., Colell, A., Marí, M., Morales, A., Fernández-Checa, J.C., 1997. Directeffect of ceramide on the mitochondrial electron transport chain leads togeneration of reactive oxygen species. Role of mitochondrial glutathione. J. Biol.Chem. 272, 11369–11377.
Gautier, J.-C., Holzhauser, D., Markovic, J., Gremaud, E., Schilter, B., Turesky, R.J.,2001. Oxidative damage and stress response from Ochratoxin A exposure inrats. Free Radic. Biol. Med. 30, 1089–1098.
Gelderblom, W.C.A., Smuts, C.M., Abel, S., Snyman, S.D., Van Der Westhuizen, L.,Huber, W.W., Swanevelder, S., 1997. Effect of Fumonisin B1 on the levels andfatty acid composition of selected lipids in rat liver in vivo. Food Chem. Toxic.35, 647–656.
Guerre, P., Larrieu, G., Burgat, V., Galtier, P., 1999. Cytochrome P450 decreases arecorrelated to increate microsomal oxidative damage in rabbit liver and primarycultures of rabbit hepatocytes exposed to AFB1. Toxicol. Lett. 104, 117–125.
Halliwell, B., Chirico, S., 1993. Lipid peroxidation: its mechanism, measurement, andsignificance. Am. J. Clin. Nutr. 57, 715S–725S.
Han, D., Loukianoff, S., McLaughlin, L., 2000. Oxidative stress indices: analyticalaspects and significance. In: Sen, C.H., Packer, L., Hanninen, O. (Eds.), Handbookof oxidants and antioxidants in exercise. Elsevier Science B.V, The Netherlands,pp. 433–484.
Harrison, L.R., Colvin, B.M., Green, J.T., Newman, L.E., Cole, J.R., 1990. Pulmonaryoedema and hydrothorax in swine produced by fumonisin B1, a toxicmetabolite of Fusarium moniliforme. J. Vet. Diagn. Invest. 2, 217–221.
Hayes, J.D., Pulford, D.J., Ellis, E.M., McLeod, R., James, R.F.L., Seidegard, J., Mosialou,E., Jernstrom, B., Neal, G.E., 1998. Regulation of rat liver glutathione-S-transferase A5 by cancer chemopreventive agents: mechanisms of inducibleresistance to aflatoxin B1. Chem. Biol. Interact. 111-112, 51–67.
Kang, Y.J., Alexander, J.M., 1996. Alterations of the glutathione redox cycle status infumonisin B1-treated pig kidney cells. J. Biochem. Toxicol. 11, 121–126.
Kellerman, T.S., Mararas, W.I.-O., Thiel, G., Gelderblom, W.C.A., Cawood, M., Coetzer,J.A.W., 1990. Leukoencephalomalacia in two horses induced by oral dosing offumonisin B1. Ondersterpoort J. Vet. Res. 57, 269–275.
Klaunig, E.J., Kamendulis, L.M., 2001. Role of oxidative stress in chemicalcarcinogenesis. In: Fuchs, J., Packer, L. (Eds.), Environmental Stressors inhealth and disease. Marcel Dekker Inc, New York, pp. 81–102.
Kouadio, J.H., Mobio, T.A., Baudrimont, I., Moukha, S., Dano, S.D., Creppy, E.E., 2005.Comparative study of cytotoxicity and oxidative stress induced bydeoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell lineCaco-2. Toxicology 213, 56–65.
Kurtz, R.S., Czuprynski, C.J., 1992. Effect of aflatoxin on in vitro production ofinterleukin-1 by bovine mononuclear phagocytes. Vet. Immunol. Immunop. 34,149–158.
Lacetera, N., Franci, O., Scalia, D., Bernabucci, U., Ronchi, B., Nardone, A., 2002.Effects on functions of ovine blood mononuclear cells for each of several fatty
757
Please cite this article in press as: Bernabucci, U., et al. Aflatoxin B1 and fumoncells. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.01.009
acids at concentrations found in plasma of healthy and ketotic ewes. Am. J. Vet.Res. 63, 958–962.
Lakritz, J., Leonard, M.J., Eichen, P.A., Rottinghaus, G.E., Johnson, G.C., Spiers, D.E.,2002. Whole-blood concentrations of glutathione in cattle exposed to heatstress or a combination of heat stress and endophyte-infected tall fescue toxinsin controlled environmental conditions. Am. J. Vet. Res. 63, 799–803.
Leal, M., Shimada, A., Ruiz, F., de Mejia, E.G., 1999. Effect of lycopene on lipidperoxidation and glutathione-dependent enzymes induced by T-2 toxin in vivo.Toxicol. Lett. 109, 1–10.
Lim, C.W., Parker, H.M., Vesonder, R.F., Haschek, W.M., 1996. Intra-venousfumonisin B1 induces cell proliferation and apoptosis in the rat. Nat. Toxins 4,34–41.
Marnett, L.J., 1999. Lipid peroxidation—DNA damage by malondialdehyde. Mutat.Res. 424, 83–95.
Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52, 711–760.Morales, A., García-Ruiz, C., Miranda, M., Marí, M., Colell, A., Ardite, E., Fernández-
Checa, J.C., 1997. Tumor necrosis factor increases hepatocellular glutathione bytranscriptional regulation of the heavy subunit chain of c-glutamylcysteinesynthetase. J. Biol. Chem. 272, 30371–30379.
Müller, G., Burkert, B., Möller, U., Diller, R., Rohrmann, B., Rosner, H., Köhler, H.,2004. Ochratoxin A and some of its derivatives modulate radical formation ofporcine blood monocytes and granulocytes. Toxicology 199, 251–259.
Neldon-Ortiz, D.L., Qureshi, M.A., 1992. The effects of direct and microsomalactivated aflatoxin B1 on chicken peritoneal macrophages in vitro. Vet.Immunol. Immunopathol. 31, 61–76.
Pestka, J.L., Bondy, G.S., 1990. Alteration of immune function following dietarymycotoxin exposure. Can. J. Physiol. Pharmacol. 68, 1009–1016.
Pugh, G.W., Richard, J.L., Kpecky, K.E., McDonald, T.J., 1984. Effect of aflatoxiningestion on the development of Moraxella bovis infectious bovinekeratoconjunctivitis. Cornell Vet. 74, 96–110.
Rumora, L., Domijan, A.-M., Zanic Grubišic, T., Peraica, M., 2007. Mycotoxinfumonisin B1 alters cellular redox balance and signalling pathways in rat liverand kidney. Toxicology 242, 31–38.
SAS, 1999. SAS user’s guide statistics 8th version. SAS Inc., Cary, NC.Šegvic-Klaric, M., Pepeljnjak, S., Domijan, A.-M., Petrik, J., 2006. Lipid peroxidation
and glutathione levels in porcine kidney PK15 cells after individual andcombined treatment with fumonisin B1, beauvericin and ochratoxin A. BasicClin. Pharmacol. Toxicol. 100, 157–164.
Selkirk, J.K., Croy, R.G., Whitlock Jr., J.P., Gelboin, H.V., 1975. In vitro metabolism ofbenzo(a)pyrene by human liver microsomes and lymphocytes. Cancer Res. 35,3651–3655.
Shen, H.M., Shi, C.Y., Shen, Y., Ong, C.N., 1996. Detection of elevated reactive oxygenspecies level in cultured rat hepatocytes treated with aflatoxin B1. Free Radic.Biol. Med. 2, 139–146.
Sivanesan, D., Begum, V.H., 2007. Preventive role of Gynandropsis gynandra L.,against aflatoxin B1 induced lipid peroxidation and antioxidant defencemechanism in rat. Indian J. Exper. Biol. 45, 299–303.
Smith, J.E., Solomons, G., Lewis, C., Anderson, J.G., 1995. The role of mycotoxins inhuman and animal nutrition and health. Nat. Toxins 3, 187–192.
Stockmann-Juvala, H., Mikkola, J., Naarala, J., Loikkanen, J., Elovaara, E., Savolainen,K., 2004. Oxidative stress induced by fumonisin B1 in continuous human androdent neural cell cultures. Free Radic. Res. 38, 933–942.
Taranu, I., Marin, D.E., Bouhet, S., Pascale, F., Bailly, J.-D., Miller, J.D., Pinton, P.,Oswald, I.P., 2005. Mycotoxin Fumonisin B1 alters the cytokine profile anddecreases the vaccinal antibody titer in pigs. Toxicol. Sci. 84, 301–307.
Towner, R.A., Qian, S.Y., Kadiiska, M.B., Mason, R.P., 2003. In vivo identificationof aflatoxin-induced free radicals in rat bile. Free Radic. Biol. Med. 35,1330–1340.
Vaca, C.E., Fang, J.-L., Mutanen, M., Valsta, L., 1995. 32P-postlabeling determinationof DNA adducts of malonaldehyde in humans: total white blood cells and breasttissue. Carcinogenesis 16, 1847–1851.
Vaca, C.E., Wilhelm, J., Hartwig, A., Harms-Ringdahl, M., 1988. Interaction of lipidperoxidation products with DNA. A review. Mutat. Res. 195, 137–149.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T.D., Mazur, M., Telser, J., 2007. Freeradicals and antioxidants in normal physiological functions and human disease.Int. J. Biochem. Cell Biol. 39, 44–84.
Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metalsand antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160,1–40.
van den Berg, J.J.M., Op den Kamp, J.A.F., Lubin, B.H., Roelofsen, B., Kuipers, F.A.,1992. Kinetic and site specificity of hydroperoxide-induced oxidative damage inred blood cells. Free Radic. Biol. Med. 12, 487–498.
Verma, R.J., 2004. Aflatoxin cause DNA damage. Int. J. Hum. Genet. 4, 231–236.Wang, M.Y., Liehr, J.G., 1995. Lipid hydroperoxide-induced endogenous DNA
adducts in hamsters: possible mechanism of lipid hydroperoxide-mediatedcarcinogenesis. Arch. Biochem. Biophys. 316, 38–46.
Wogan, G.N., 1999. Aflatoxin as a human carcinogen. Hepatology 30, 217–221.Yin, J.J., Smith, M.J., Eppley, R.M., Page, S.W., Sphon, J.A., 1998. Effects of fumonisin
B1 on lipid peroxidation in membranes. Bioch. Biophys. Acta 1371, 134–142.
isin B1 affect the oxidative status of bovine peripheral blood mononuclear
