investigation of in vivo toxicity of hydroxylamine sulfate and the efficiency of intoxication...

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Food and Chemical Toxicology xxx (2013) xxx–xxx

FCT 7444 No. of Pages 6, Model 5G

22 July 2013

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Investigation of in vivo toxicity of hydroxylamine sulfateand the efficiency of intoxication treatment by a-tocopherolacetate and methylene blue

0278-6915/$ - see front matter � 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.fct.2013.07.024

Abbreviations: HA, hydroxylamine; HAS, hydroxylamine sulfate; ESR, ElectronSpin Resonance; MetHb, methaemoglobin; TBARs assay, thiobarbituric acid reactivesubstances assay; DTNB, Ellman’s reagent 5,50-dithiobis-(2-nitrobenzoic acid); LPO,lipid peroxidation.⇑ Corresponding author. Tel./fax: +380 44 5269347.

E-mail addresses: [email protected] (M.G. Prodanchuk), [email protected] (A.K. Tsakalof).

1 Tel.: +30 2410 685580.

Please cite this article in press as: Prodanchuk, M.G., et al. Investigation of in vivo toxicity of hydroxylamine sulfate and the efficiency of intoxicatioment by a-tocopherol acetate and methylene blue. Food Chem. Toxicol. (2013), http://dx.doi.org/10.1016/j.fct.2013.07.024

Mykola G. Prodanchuk a,⇑, Aristidis M. Tsatsakis b, Georgiy M. Prodanchuk a, Andreas K. Tsakalof c,1

a Ministry of Health, Institute of Ecohygiene and Toxicology, 6, Heroiv Oborony Str., UA-252022 Kiev, Ukraineb Centre of Toxicology Sciences and Research, Division of Morphology, Medical School, University of Crete, Voutes, Heraklion 71003, Crete, Greecec Laboratory of Chemistry, Faculty of Medicine, University of Thessaly, Larissa, Greece

a r t i c l e i n f o

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Article history:Available online xxxx

Keywords:Hydroxylamine sulfateHematotoxicityOxidative stressa-TocopherolMethylene blueMechanism of toxicity

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a b s t r a c t

Objectives: Investigation of hydroxylamine sulfate toxicity mechanism in vivo and estimation of a-tocopherol acetate and methylene blue efficiency in poisoning treatments.Methods: In vivo experiments were conducted on 102 Wistar Han rats. The experiments investigated thehematotoxic and oxidative stress effects of hydroxylamine sulfate in acute and subacute toxicity treat-ment of animals. Electron Spin Resonance was used for quantitative determination of blood and liver tis-sue parameters alterations after intoxication. The osmotic fragility of erythrocytes, lipid peroxidationintensity and level of SH-groups in liver of rats were determined by established biochemical assays.Results: Hydroxylamine sulfate cause an acute hematotoxicity and oxidative stress in vivo as demon-strated by the appearance of free oxidized iron in blood, reduced glutathione content and increased lipidperoxidation in liver. The experimental studies showed the formation of Hb–NO, MetHb in erythrocytesand as well of stable complex of reduced iron (Fe2+) with hydroxylamine sulfate. Methylene blue treat-ment does not reduce the Hb–NO or MetHb levels in intoxicated animals while administration of a-tocopherol acetate reduces substantially lipid peroxidation.Conclusions: Oxidative stress is a key mechanism of acute hematotoxicity caused by hydroxylamine sul-fate. Methylene blue is not suitable antidote in case of hydroxylamine intoxication.

� 2013 Published by Elsevier Ltd.

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1. Introduction

Hydroxylamine (HA) and hydroxylamine sulfate (HAS) arewidely used as intermediates in synthesis of pharmaceuticals, pes-ticides, dyes, caprolactam and other organic compounds. As typicalmethemoglobin forming agents, HA and HAS are under close atten-tion of many researchers because of their unique biological effects.In comparison to other xenobiotics generating methemoglobinthese agents are characterized by faster and more intensive toxicmethemoglobinemia development expressed by hemolyticanemia, sulfohemoglobinemia and various systemic disorders

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(Pacheco et al., 2011; Bordin and Fiore, 2010; Boutrin and Wang,2012; Gross, 1985; Grossman et al., 1992; Mc Millan et al.,1995). Despite numerous studies, the mechanisms of formationof toxic methemoglobinemia and hemolytic anemia in case of HAand HAS intoxications are not completely explained and this makesthe development of effective therapy troublesome (Gharahbaghian,2009; Lim and Tan, 2009; Roque, 2008; Kankuri et al., 2001). Therole of reactive oxygen forms, nitric oxide and free blood iron information of hemo- and cytotoxic effects caused by HAS intoxica-tion needs further investigation. It is still difficult to explain theremaining metabolic hypoxia and development of systemic disor-ders in patients after reduction of methemoglobin (MtHb) andregeneration of hemoglobin (Hb) levels (Rassaf et al., 2003; Reiteret al., 2003). The effectiveness of the methylene blue antidote anda-tocopherol acetate for the treatment of hydroxylamine sulfatepoisoning remains unexplored (Dotsch et al., 2000; Eyer et al.,2003). The above issues need to be clarified in order to developapproved methods of diagnostics and justified clinical protocolsfor the treatment of such intoxications.

n treat-

http://dx.doi.org/10.1016/j.fct.2013.07.024

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2. Materials and methods

2.1. Animals and administration protocol

One hundread two healthy Wistar Han rats (weighting between 200 and 250 geach) were used in 4 different in vivo studies. The animals were randomly selectedand housed in T4 polycarbonate cages (five rats per cage) with steel wire tops anddechlorinated paper bedding. They were maintained in barrier facility with con-trolled atmosphere, 12-dark/12-light cycle, 22 ± 2 �C temperature ranges and 50%humidity with free access to pelleted feed and fresh purified filtered water. The ani-mals were supplied with standard Altromin 1326 dry food pellets commerciallyavailable from Altromin Spezialfutter GmbH & Co. KG. They were allowed to accli-matize for 5 days before the start of the treatment. Institutional Bioethical Commit-tee approved the above mentioned studies.

2.1.1. In vivo studies included

1. Investigation of hematotoxic effect of HAS acute poisoning.

Twenty four rats were treated with hydroxylamine sulfate in one dose (400 mg/kg) by oral gavage. The exposed animals were divided in 4 groups of 6 animals andthese groups were sacrificed after 0.5; 1; 3 and 24 h after exposure. The controlgroup of 6 rats was administered water in the same manner as in the treatmentgroups and sacrificed after 24 h period in CO2 chamber.

2. Investigation of hematotoxic effect of HAS subacute poisoning.

Six rats were treated with hydroxylamine sulfate daily with 80 mg/kg dose byoral gavadge for 12 days. Six animals of the control group were treated with waterin the same manner as in the treatment group. All animals were sacrificed on 12thday in CO2 chamber.

3. Investigation of methylene blue efficiency in cases of acute poisoning caused bysodium nitrite and hydroxylamine sulfate.

Thirty Wistar Han rats were randomly divided into 5 groups of 6 rats. The firstgroup was treated with sodium nitrite 60 mg/kg subcutaneously. The second groupwas administered sodium nitrite subcutaneously in dose 60 mg/kg and 50 min afterwith 1% methylene blue in dose 20 mg/kg intraperitoneal. The third group was trea-ted with hydroxylamine sulfate in dose 80 mg/kg. The fourth group was adminis-tered 80 mg/kg of hydroxylamine sulfate and 1% methylene blue in dose 20 mg/kg 50 min after. Control group was treated with water in the same manner as inthe treatment groups. All animals were sacrificed 2 h after exposure in CO2

chamber.

4. Investigation of a-tocopherol acetate efficiency in case of acute poisoning byhydroxylamine sulfate.

Three groups of 6 animals were formed from randomly selected 18 Wistar Hanrats. The first group was treated with hydroxylamine sulfate in dose 400 mg/kg. Thesecond group was treated with hydroxylamine sulfate in dose 400 mg/kg and with5% sodium bicarbonate subcutaneously and 90 min after was administered 10%alpha-tocopheryl acetate sunflower oil solution in dose 5 mg/kg per os. Controlgroup was treated with water in the same manner as in the treatment groups. Allanimals were sacrificed 3 h after exposure in CO2 chamber.

2.2. Determination of blood and liver parameters using Electron Spin Resonancespectroscopy

Samples of blood and tissue were prepared using a press-form. The press-formis a teflon cylinder with the following dimensions: 65 mm in height, and 15 mm indiameter with a recess of 4.5 mm in diameter and 60 mm in depth filled with asample.

The samples were frozen in liquid nitrogen (77 K) and removed from the pressform after complete cooling. The analysis was performed using the Varian E-109EPR spectrometer. The instrument settings for whole blood were as follows: micro-wave power – 5 mW, magnetic induction – 200 mT, frequency range – 400 mT,response time – 2 s., scan time – 16 min, temperature – 77 K. A rubin was usedas an internal standard.

The instruments settings for detection of MetHb and Hb–NO in blood were:microwave power – 8 mW (for transferrin) and 2 mW (for ceruloplasmin), fre-quency range – 100 mT, modulation amplitude – 0.8 mT, response time – 0.5 s(for transferrin) and 1 s (for ceruloplasmin), operating frequency of the spectrome-ter 9075–9095 MHz, scan time – 2 min (for transferrin) and 4 min (for ceruloplas-min). The estimation was performed by comparison of the test sample spectrumto a reference sample.

Please cite this article in press as: Prodanchuk, M.G., et al. Investigation of in vivment by a-tocopherol acetate and methylene blue. Food Chem. Toxicol. (2013

Liver tissue parameters were detected with the following instruments settings:microwave power – 5 mW, magnetic induction – 260–360 mT, frequency range –100 mT, modulation amplitude – 0.8 mT, response time – 0.5 s, operating frequencyof the spectrometer 9075–9095 MHz, scan time – 4 min. The estimation of ESRspectroscopy parameters detected in blood and liver of Wistar Han rats was per-formed by comparison of ESR signal amplitude with a G-factor of control and inves-tigated sample tissues. The results are measured in relative units.

2.3. Determination of lipid peroxidation intensity and level of SH-groups in liver ofWistar Han rats

The TBARs assay was used for measuring the peroxidation intensity. This isbased on the reaction of TBA (thiobarbituric acid) with the end product of lipid per-oxidation–malondialdehyde. Level of the lipid peroxidation was measured in wholered cells.

The concentration of –SH groups was measured by Ellman’s method. The livertissue homogenate was mixed with 10% SDS and H2O and that mixture was incu-bated in phosphate buffer with the solution of DTNB in sodium citrate. After 30-min incubation, the spectrophotometric absorption was measured at 412 nm.

2.4. Determination of osmotic fragility of erythrocytes

Blood samples were collected from 6 animals of experimental subacute dosingand control groups. Erythrocytes resistance against lysis was evaluated as a result ofthe osmotic pressure changes of their surrounding media. The 25 ll of erythrocytesample was added to each of a series of 2.5 ml saline solutions containing 0.3–0.9%of NaCl. After gentle mixing and standing for 15 min at room temperature, theerythrocyte suspensions were centrifuged at 5000 rpm for 5 min. The absorbanceof the supernatant was measured at 540 nm. The absorbance percentage releasedhemoglobin was expressed as percentage absorbance of each sample in correlationto a completely lysed sample prepared by diluting of packed cells of each type with1.5 ml of distilled water.

3. Results

3.1. Investigation of HAS hematotoxic effect in acute toxicity treatmentof animals

The data obtained using the Electron Spin Resonance spectros-copy method revealed that in group of animals treated withhydroxylamine sulfate in dose 400 mg/kg the level of MetHb ex-ceeded 56 mg/l (almost 50% of total Hb in blood) in 30 min afterexposure (Table 1). High anisotropic singlet signal with triplet finesplitting (g = 2.03) was also found in this group of animals. Accord-ing to its parameters and form, this signal is absolutely identical tothe signal of hemoglobin nitrosyl complex formed by hemoglobininteraction (Fig. 1). This signal is not observed in ESR spectrumof the control group. Thus, we assume that abovementioned signalis the signal of Hb–NO complex, which appeared in blood of exper-imental animals as a result of poisoning by hydroxylamine sulfate

The level of this Hb–NO complex has decreased 4.5-fold in 1 hafter exposure, in comparison to MetHb level which has decreasedonly by 3-fold during the same period of time. MetHb level showed30-fold decrease from the maximum value and approached thephysiological value of control group in 24 h after exposure (Table1) in contrast to nitrosyl hemoglobin level that has decreased onlyby 10 times and still remained high enough to affect blood oxygentransporting function.

Hb–NO complex contains heme iron in a oxidized state (Fe3+)during the first few minutes, then it quickly reduces to Fe2+, but be-cause of a strong covalent bond of the adduct, as in case of MetHb,it loses its oxygen binding capacity. Obviously this disorder plays amain role in the mechanism of acute poisoning by hydroxylaminesulfate.

A severe cyanosis of extremities, rapid breathing and heart rate,hypodynamia and movement discoordination were observedamong the experimental group of animals in 1 h after exposure.The signs of above mentioned symptoms remained present evenin 24 h after exposure by hydroxylamine sulfate. This could be ex-plained by the presence of a large amount of Hb–NO in blood as thelevel of MetHb at the same time was in normal range.

o toxicity of hydroxylamine sulfate and the efficiency of intoxication treat-), http://dx.doi.org/10.1016/j.fct.2013.07.024


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Table 1ESR spectroscopy parameters detected in blood and liver of Wistar Han rats after single exposure by hydroxylamine sulfate in dose 400 mg/kg (M ± m; n = 6).

Values In control group In experimental groups, period after exposure (h)

0.5 1 3 24

In bloodMetHb (g/l) 1.21 ± 0.11 56.2 ± 8.1* 17 ± 3* 3.3 ± 0.5* 1.9 ± 0.2*

Hb–NO (g/l) 0 23 ± 6* 5.1 ± 1.3* 3.8 ± 1.0* 2.3 ± 0.5*

Fe3+ transferrin (mg/l) 0.48 ± 0.11 0.52 ± 0.13 0.50 ± 0.10 0.47 ± 0.10 0.51 ± 0.14Fe3+ labile (mg/l) 0 0 0 0.10 ± 0.08* 0.85 ± 0.39*

In liverCYP450 (rel. units) 1.01 ± 0.21 0.89 ± 0.18 1.15 ± 0.21 0.97 ± 0.18 0.88 ± 0.19Mn2+-centres (rel. units) 1.01 ± 0.11 1.00 ± 0.17 0.97 ± 0.07 1.00 ± 0.10 0.77 ± 0.13*

Cu2+-centres (rel. units) 1.02 ± 0.04 1.04 ± 0.12 1.00 ± 0.08 1.08 ± 0.16 0.68 ± 0.08*

NO-complexes (rel. units) 1.00 ± 0.26 1.61 ± 1.03 2.03 ± 0.87* 1.68 ± 0.26* 0.87 ± 0.61Mo7+-centres (rel. units) 1.00 ± 0.20 2.40 ± 0.80* 2.80 ± 0.40* 2.80 ± 0.86* 2.20 ± 0.60*

FeS-centres (rel. units) 1.00 ± 0.12 1.04 ± 0.15 1.08 ± 0.10 0.94 ± 0.08 0.96 ± 0.06Q-free radicals (rel. units) 1.00 ± 0.10 1.10 ± 0.12 1.14 ± 0.15 1.10 ± 0.09 1.20 ± 0.09*

n – Number of animals in group, M – mean value, m – mean square error.* Statistically significant difference with control group, p < 0.05.

Fig. 1. ESR signals of nitrosyl hemoglobin (Hb–NO): (A) formed as a result ofhemoglobin interaction with NO as a result of animal treatment with sodium nitriteand (B) found in blood of animals, treated with hydroxylamine sulfate.

Table 2Blood parameters of control and experimental groups treated with hydroxylaminesulfate during 12 days in dose 80 mg/kg (M ± m; n = 6).

Values In control group In experimental group

Hb (g/l) 114.2 ± 16.1 76.2 ± 7.1*

MetHb (g/l) 0.9 ± 0.1 1.2 ± 0.4Hb–NO (g/l) 0 1.7 ± 0.5*

OxyHb (%) 88 ± 1.1 74 ± 3.1*

DeoxyHb (%) 0.5 ± 0.1 8.2 ± 1.5*

Fe3+-labile (mg/l) 0 0.8 ± 0.2*

n – Number of animals in group, M – mean value, m- mean square error.* Statistically significant difference with control group, p < 0.05.

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Significant concentrations of free ferric iron were observed inblood of experimental animals being under the influence ofhydroxylamine sulfate for 3 and 24 h periods of exposure (Table 1).

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Fig. 2. ESR signal, detected in liver tissue of rats from experimental group treatedwith sodium nitrite (A) and with hydroxylamine sulfate (B). Signal with g = 2.03 –nitrosyl complex with liver proteins; g = 2.25 – cytochrome P-450; g = 1.94 – iron-sulfur proteins.

3.2. Investigation of HAS hematotoxic effect in subacute toxicitytreatment of animals

The level of MetHb was close to normal range of control group,but the ESR signal of nitrosyl hemoglobin, which was absent incontrol animals, was observed in rats treated with hydroxylaminesulfate in dose 80 mg/kg for 12 days (Table 2, Fig. 2). After 12 daysof exposure the level of labile oxidized iron was increased in com-parison to control group and close to the level observed in acutedosing after 24 h (Tables 1 and 2).

It was found that level of hemoglobin in blood decreased by 33%(Table 2) and resistance of erythrocytes also notably decreased inexperimental group of animals that was treated with hydroxyl-amine sulfate in dose 80 mg/kg during 12 days (Table 3). Thesechanges indicate the possibility of development of hemolytic ane-mia in treated animals.

Another finding in the experimental group of animals (OxyHblevel decreased by 16%, deoxyHb level increased by 16.4 times)indicates to a limited ability of hemoglobin to transport oxygenmainly due to the formation of deoxyHb. The ESR signal registedin blood of experimental animals is typical for nitrosyl hemoglobin


presence and apparently can be explained by the fact that hydrox-ylamine sulfate in cells is biotrasformed with the release of NO.Such a conclusion was made by different researchers who studiedthe effect of hydroxylamine and its derivatives (Bradshaw et al.,1997; Gharahbaghian and Massoudian, 2009).

However, the changes of MetHb and Hb–NO levels in blood,caused by treatment with hydroxylamine sulfate, differs from thatobserved under influence of NO and its donors. In the latter case adecay of nitrozyl complex in heme is the main reason for formationof MetHb and precedes it.



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Table 3The resistance of erythrocytes in experimental group of animals treated withhydroxylamine sulfate in dose 80 mg/kg during 12 days. (M ± m; n = 6).

NaCI concentrations in medium forincubation of erytrocytes (%)

Number of hemolyzederythrocytes (%)

In controlgroup

Inexperimentalgroup

0.900 0 00.600 0 00.550 0 00.525 0 1.3 ± 0.1*

0.500 4.2 ± 0.6 7.7 ± 0.9*

0.475 11.2 ± 2.1 13.3 ± 2.10.450 33.2 ± 4.2 39.3 ± 4.10.400 79.3 ± 9.1 75.2 ± 2.20.300 100 100

n – Number of animals in group, M – mean value, m – mean square error.* Statistically significant difference with control group, p < 0.05.

Table 5Effect of methylene blue on MetHb and Hb–NO levels in blood of animals, exposed tohydroxylamine sulfate and sodium nitrite (M ± m, n = 5).

Group of animals MtHb (g/l) Hb–NO (rel. units)

1. Control 1.1 ± 0.2 02. Sodium nitrite 28.2 ± 4.1 180.3 ± 40.23. Sodium nitrite + methylene blue 7.3 ± 2.4* 190.2 ± 60.34. Hydroxylamine sulfate 22.3 ± 5.1 310.3 ± 70.25. HAS + methylene blue 20.2 ± 6.1 290.2 ± 60.1

n – Number of animals in group.* Statistically significant difference with control group, p < 0.001.



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3.3. Investigation of methylene blue efficiency in cases of acutepoisoning caused by sodium nitrite and hydroxylamine sulfate

In acute poisoning with such well-known donor of NO as so-dium nitrite a much more intensive ESR signal typical for Hb–NOcomplex of non-heme iron is observed in liver tissue in comparisonto that observed after single dose administration of hydroxylaminesulfate. Also, a significant reduction of magnitude of ESR signalstypical for CYPs, as well as for Mn2+, Cu2+ and reduced iron-sulfur(mitochondrial) centers, which are direct targets for free NO, is ob-served after adminstration of sodium nitrite.

The ESR signal of coenzyme Q free radicals is also significantlydecreased, as it was confirmed by other researchers (Kehl, 1997;Palmen Nicole and Evelo Chris, 1998). A completely differentdynamics and direction of changes in abovementioned parameterswere observed in the liver of rats treated with hydroxylamine sul-fate. In particular, the ESR signal of Mo7+ center, typical for xan-thine oxidase, had rapid increase and remained at such level fornext 24 h. The formation of H2O2 and superoxide is usually in-creased during activation of xanthine reaction. The amount ofcoenzyme Q free radicals increases after 24 h as well, which indi-cates a formation of reactive oxygen species. The height of ESRsignal amplitude for Mn2+ and Cu2+ centers is reduced by 23%and 32%. These elements are included in the active sites structureof superoxide dismutase and ceruloplasmin. Thus, the abovechanges indicate a weakening of antioxidant protection due to areduction of superoxide dismutase and ceruloplasmin amounts.

Indicated signs of oxidative stress are obviously consistent withthe induction by hydroxylamine sulfate of spontaneous (40%) andascorbate dependent (36–40%) levels of malondialdehyde in the li-ver, indicating the intensification of LPO. Oxidation processes inthis case are accompanied by an increased level of free SH-groupsin the liver, the main carrier of which is glutathione (Table 4). HAS

Table 4LPO parameters and level of SH-groups in liver after one oral administration of hydroxyla

Group of animals Amount of

Background

Control group 5.7 ± 1.1Experimental group time after administratin of HAS (hours) 0.5 6.2 ± 1.3

1 6.6 ± 1.13 5.7 ± 0.924 6.4 ± 1.1

n – Number of animals in group, M – mean value, m – mean square error* Statistically significant difference with control group, p < 0.05.


and sodium nitrite, both cause increasing of MetHb and hemoglo-bin nitrosyl levels in blood of experimental animals. Methyleneblue in both cases did not affect the content of Hb–NO. These re-sults clearly confirm our findings regarding the differences in themechanisms of hemoglobin interaction with nitric oxides, on theone hand, and with hydroxylamine sulfate – on the other (Table 5).

3.4. Investigation of a-tocopherol acetate efficiency in case of acutepoisoning by hydroxylamine sulfate

The results of administration of a-tocopherol acetate andsodium bicarbonate for correction of acidosis in case of acute poi-soning by hydroxylamine sulfate in dose 400 mg/kg are summa-rized in Table 6.

The significant increase of spontaneous and ascorbate depen-dent lipid peroxidation levels, by 36% and 71% respectively, isobserved after treatment of animals with hydroxylamine sulfate.

An administration of a-tocopherol acetate simultaneously withsodium bicarbonate for correction of acidosis reduces these param-eters to values even lower than in control rats.

4. Discussion

Despite the studies conducted, the mechanisms of HA and HAStoxicity leading to methemoglobinemia and hemolytic anemia arestill not clearified. (Wrobel et al., 2003; Spooren and Evelo, 1997a).A pathogenetic significance of reactive oxygen and nitrogen formsremains unknown in the genesis of hemo- and cytotoxic effects ofHAS. Experimental investigations of several authors proved thatintoxication with xenobiotic-reductants (HA, HAS, hydrazine,phenylhydrazine, etc.) is accompanied by persistent degenerativechanges in erythrocytes, hemoglobin derivatives accumulationand activation of prooxidant processes. Literature data indicatethat hydroxylamine and its compounds cause metabolic acidosisin the body (Yong and Beams, 2000; Alderton et al., 2001; Adamsand Brochwicz-Lewinski, 1999; Crane et al., 1997).

Analysis of the available literature data on the mechanisms ofhematotoxic effects of hydroxylamine and its derivatives revealedsome contradictions and a number of unresolved issues. Mostauthors are inclined to believe that the toxic methemoglobinemia

mine sulfate in dose 400 mg/kg (M ± m; n = 6).

malondialdehyde (nmole/g of tissue) SH-groups (lmole/g of tissue)

Spontaneous Ascorbate dependent

23.2 ± 2.1 351.3 ± 19 24.2 ± 2.134.3 ± 2.1* 499.2 ± 23* 31.3 ± 3.2*

33.2 ± 2.2* 478.3 ± 18* 28.2 ± 4.133.2 ± 2.1* 481.4 ± 13* 34.2 ± 3.2*

33.2 ± 2.1* 421.4 ± 26 39.2 ± 6.1*



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Table 6Parameters of lipid peroxidation in liver of animals, treated with hydroxylamine sulfate, (M ± m, n = 6).

Group of animals Amount of malondialdehyde (nmole/g of tissue)

Background Spontaneous Ascorbate dependent

1. Control 5.2 ± 0.4* 22.8 ± 0.6 380.6 ± 2.92. Hydoxylamine sulfate 7.8 ± 0.4* 42.2 ± 0.8* 558.4 ± 5.5*

3. Hydoxylamine sulfate + vit E + sodium ascorbate 4.4 ± 0.3** 7.9 ± 0.4** 44.5 ± 0.2**

n – Number of animals in group.* Statisticaly significant values, p < 0.001 comparing to control group.** Statisticaly significant values, p < 0.001 comparing to group, treated with hydroxylamine sulfate.

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and hemolytic anemia under exposure to hydroxylamine and itscompounds are due to the formation of oxidative stress, which in-cludes the formation of a number of reactive compounds (superox-ide anion, nitric oxide, peroxide, peroxynitrite, etc.). (Tang et al.,2005; Fradette et al., 2004; Spooren and Evelo, 1996, 1997a,b,1998). It is believed that reactive oxygen and nitrogen speciesand the compounds of non-radical nature interact with cell thiolsand membrane lipids, which in its turn leads to the oxidation oferythrocyte glutathione and its energy-dependent export, as wellas the oxidation of sulfhydryl groups of membranes. There are noconvincing data on the reason of persistent MetHb-formation,which is resistant to methylene blue and ascorbic acid treatment.It cannot be clearly explained what mechanisms lead to hemolyticanemia under exposure to these xenobiotics (Tang et al., 2005;Fradette et al., 2004; Spooren and Evelo, 1996, 1998; Stoianet al., 1996; Lonart and Johnson, 1998; De Sesso and Goeringer,1990; Kurian and Bajad, 2004). Conducted by us experimentalin vivo studies in rats using ESR techniques showed that the admin-istered HAS creates a strong adduct with the heme iron. With re-gards to ESR spectral characteristics this transformed adduct ofreduced iron (Fe2+) with HAS is absolutely identical to the nitrozylcomplex formed by the interaction of hemoglobin with NO. Thepresence of a strong adduct of HAS with iron (as Fe3+ and Fe2+) inerythrocytes, obviously, is one of the main reason of oxygen trans-porting function failure and persistent structural and metabolicchanges that lead to a decrease in resistance of these cells. Anadministration of HAS in rats cause the state of oxidative stress,as indicated by the changes in ESR signals typical for xanthine oxi-dase and Q-ubiquinon free radicals and reduction of those radicalsrelated to superoxide dismutase and ceruloplasmin. The oxidativestress is also characterized by decreased content of SH-groups, thepresence of labile oxidized iron in the blood and the intensificationof lipid peroxidation (LPO) in the liver, which facilitates the reali-zation of HAS hematotoxic effects. The intracellular formation offree NO in erythrocytes makes some contribution to the formationof HAS hematotoxic violations, but apparently plays not majorpathogenic role, as indicated by a slight increase in the total levelof nitrite and nitrate in blood serum. Perhaps intracellularlyformed NO is involved in the formation of stable iron nitrosyl com-plexes in erythrocytes, and therefore the intensive growth of thetotal content of nitrite and nitrate levels in serum is not observed.The experiment on rats showed that HAS poisoning of animalsaccompanied by increased spontaneous and ascorbic-dependentperoxidation by 71% and 36% respectively. The administration ofa-tocopherol acetate (vitamin E) with simultaneous correction ofmetabolic acidosis by sodium bicarbonate reduces the intensityof LPO to a level lower than in control rats.

The use of antidotes for treatment of poisoning by methemoglo-bin-forming substances is generally accepted in clinical practice.Methylene blue is widely used in such cases. It accelerates theenzymatic reduction of methemoglobin by NADPH-methemoglo-bin reductase and also reduces to leucomethylene blue that, inits turn, reduces methemoglobin. This fact prompted us to evaluatethe effectiveness of methylene blue administration in HASpoisoning.


However, our experimental data indicate the inefficiency ofmethylene blue in the reduction of Hb–NO complex formed asthe result of HA/HAS poisoning and lead to the conclusion thatmethylene blue can be used as antidote in HA/HAS intoxication.

Also, the conclusions were made regarding the appropriatenessof further administration of a-tocopherol acetate with sodiumbicarbonate for correction of acidosis in the treatment of acuteHAS poisoning.

5. Conclusions

Summarizing the results of this study, we came to the followingconclusions:

The experimental studies in rats using ESR techniques showedpossible mechanism of the formation of NO in erythrocytes andfirmly bound adduct of heme iron, which was originally in the oxi-dized form (MetHb-HAS) and resistant to reducing agents such asmethylene blue.

As per ESR spectral characteristics, newly formed complex ofreduced iron (Fe2+) with HAS adduct is completely identical to anitrosyl complex, formed by the interaction of hemoglobin withNO and its donors.

The presence of strong HAS adducts with iron (in a form of Fe3+,Fe2+) in erythrocytes deprives its oxygen-transport function andleads to persistent metabolic and physiological changes, as indi-cated by reduction in osmotic resistance of erythrocytes.

By the results of experimental studies in vivo in rats, the appli-cation of a-tocopherol acetate with sodium bicarbonate for back-ground correction of acidosis is justified in acute poisoning withhydroxylamine sulfate.

The verification of the hypothesis on possible mechanisms ofhydroxylamine sulfate action in body requires further in vitrostudies.

Conflict of Interest

The authors declare that are no conflicts of interest.

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investigation of in vivo toxicity of hydroxylamine sulfate and the efficiency of intoxication...

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