Histological and biochemical features of theliver in juvenile male rats following subchronicacrlyamide exposure

Akrilamid (CASR No. 79-06-1) predstavlja veoma reaktivni, hidrosolubilni monomer za koji se smatra da ima toksične i potencijalno kancerogene efekte po zdravlje ljudi. Štetne posledice akrilamida i njegovog još reaktivnijeg metabolita, glicidamida, su dokazane kod eksperimentalnih životinja i podrazumevale su neurotoksičnost, genotoksičnost i kancerogenost. Epidemiološke studije rađene na ljudima pokazale su da akrilamid izaziva neurotoksične efekte, dok se genotoksičnost i kancerogenost još smatraju potencijalnim efektima, a zasnivaju se na podacima koji su dobijeni u okviru istraživanja na laboratorijskim životinjama. Njegove štetne posledice na jetru, posebno kod mladog organizma, još uvek nisu dovoljno istražene. Akrilamid se spontano formira u hrani koja je bogata ugljenim hidratima, tokom termičke obrade namirnica na visokim temperaturama. Ovaj monomer se formira tokom tzv. neenzimatske Mallard-ove reakcije, kojom se dobijaju smeđe komponente u hrani. U namirnicama ovaj monomer se formira reakcijom između redukujućih šećera (uglavnom glukoze ili fruktoze) i aminokiseline (dominantno asparagina). Imajući na umu da je jetra centralni organ za metabolizam akrilamida, ovo istraživanje je imalo za cilj da ispita glavne histološke i biohemijske promene na jetri juvenilnog organizma pacova, nakon njegove subhronične ekspozicije akrilamidu. Istraživanje je rađeno na 3 eksperimentalne grupe peripubertalnih/juvenilnih mužjaka Wistar pacova, od kojih su dve bile tretirane vodenim rastvorom akrilamida u dozi od 25 ili 50 mg/kg telesne mase, dok je treća grupa služila kao kontrola i primala destilovanu vodu. Životinje su bile tretirane oralno, putem gavaže, 5 dana nedeljno, tokom 3 nedelje. Nakon 24 h od poslednjeg tretmana, životinje su uvedene u etarsku anesteziju i dekapitovane, a zatim su prikupljeni krv i uzorci tkiva jetre. Tkivo jetre je uzeto iz srednjeg lobusa, fiksirano u 10% neutralnom puferisanom formalinu tokom 24 h i obrađeno prema standardnom protokolu za parafinsko kalupljenje. Ukalupljeni uzorci jetre su zatim isečeni na serijske preseke tkiva debljine 5 µm, a zatim bojeni histohemijskim i imunohistohemijskim metodama. Uzorci krvi su pripremljeni za serološku analizu. Histološka analiza preseka bojenih hematoksilin-eozin (H&E) metodom nije zabeležila prisustvo značajnih razlika u opštoj arhitekturi jetre i njenoj lobularnoj organizaciji među eksperimentalnim grupama. Stereološka analiza je ukazala na mikrostrukturne promene kod hepatocita i jetrinih sinusoida. Rezultati sugerišu, na dozno-zavisno povećanje volumena hepatocita, njihove citoplazme i nukleusa, i doznozavsino smanjenje volumena sinusoida, u odnosu na kontrolne uzorke jetre. Analiza glikogena je rađena na presecima jetre bojenim metodom Periodic acid– Schiff-a (PAS), gde se uočilo smanjenje količine glikogena u grupi tretiranoj nižom dozom akrilamida, dok je u grupi tretiranoj većom dozom uočena njegova akumulacija, u odnosu na kontrolne životinje. Imunopozitivnost hepatocita na marker proliferacije, Ki-67, bila je smanjena u grupi pacova tretiranoj nižom dozom, a bila povećana u grupi tretiranoj većom dozom akrilamida pri komparaciji sa kontrolom. Stereološki nalazi su potvrdili inicijalnu histološku analizu. Imunopozitivnost hepatocita na marker apoptoze, Caspase 3, je bila smanjena kod obe grupe životinja tretiranih akrilamidom u odnosu na kontrolu. Nasuprot tome, imunopozitivnost neparenhimskih ćelija jetre, pretežno Kupffer-ovih ćelija, je bila uvećana u obe tretirane grupe pri komparaciji sa kontrolom. Imunopozitivnost Kupffer-ovih ćelija na marker CD68 je bila smanjena u uzorcima jetre kod oba tretmana akrilamidom u odnosu na kontrolu. Populacija mastocita, prikazana toluidine-blue (TB) metodom bojenja, bila je uvećana kod obe grupe pacova tretiranih akrilamidom u poređenju sa kontrolom. Povećanje brojnosti ovih ćelija je bilo posebno prominentno kod njihove degranulisane subpopulacije. Stereološka analiza je potvrdila histološke nalaze. Serumska analiza je pokazala uvećanu aktivnost aspartat aminotrasferaze (AST) i smanjenu aktivnost alanin aminotrasferaze (ALT) kod obe grupe životinja tretiranih akrilamidom u odnosu na kontrolu. Aktivnost alkalne fosfataze (ALP) je bila uvećana u grupi tretiranoj nižom dozom, a smanjena u grupi tretiranoj većom dozom akrilamida, u odnosu na kontrolu. Vrednosti koncentracije ukupnih serumskih proteina kao i koncentracije C reaktivnog proteina (CRP) nisu pokazale značajnije promene među eksperimentalnim grupama. Oba akrilamidna tretmana su izazvala gubitak telesne mase kod tretiranih pacova, u odnosu na kontrolne životinje. Postojeći podaci ukazuju prominentni hepatotoksični potencijal akrilamida koji može poremetiti mikrostrukturne osobine i funkcionalni status hepatocita kod jetre mladog organizma. Akrilamid može značajno poremetiti funkcionalnost jetre, obzirom da se promene na celularnom nivou mogu relativno brzo odraziti na nivo tkiva, a kasnije ugroziti i homeostazu celog organizma.Acrylamide (CASR No. 79-06- 1) is highly reactive, water-soluble monomer which is considered as toxic and potentially cancer causing chemical to humans. Adverse health effects regarding acrylamide and its more reactive metabolite,glycidamide, were detected in experimental animals, and included neurotoxicity, genotoxicity, and carcinogenicity. Human epidemiological studies claim that acrylamide has neurotoxic effects, while genotoxicity and carcinogenicity are considered as potential human health risks only on the basis of animal studies. Its harmful effects on the liver, especially in a young organism, are still to be elucidated. Acrylamide is spontaneously formed in carbohydrate-rich food during high-temperature processing. It is formed during heat-induced non-enzymatic reaction, also known as the Maillard browning reaction, between reducing sugars (glucose and fructose), and free amino acids (mainly asparagine). Having in mind that acrylamide metabolism takes place in a liver, the study aimed to investigate the main histological and biochemical changes in the liver of juvenile rat following subchronic acrylamide intoxication. Study was performed on peripubertal/juvenile male Wistar rats, divided in 3 experimental groups, two of which were treated with acrylamide in doses of 25 or 50 mg/kg of body weight, while the third group served as the control and received distilled water. Animals were treated orally, via gavage, 5 days a week, during 3 weeks. Animals were anesthetized by ether inhalation and decapitated 24 hrs after the last treatment. Liver tissue was sampled from the middle lobe, fixed in 10% neutral buffered formalin for 24 hrs, routinely processed for paraffin embedding and cut into 5-µm thick serial sections for subsequent histochemical and immunohistochemical staining.Blood samples were collected for subsequent biochemical analysis . Histological examination of haematoxylin and eosin (H&E) stained sections did not point to any major alteration in main in liver lobular architecture or organization among the experimental groups. Stereological analysis revealed a microstructural changes in hepatocytes and liver sinusoids. The analysis detected a dose-dependant increase in the volume of hepatocytes, their cytoplasm and nuclei, and dose-dependant decrease in the volume of liver sinusoids compared to the control, respectively. Glycogen analysis was performed on Periodic acid–Schiff (PAS) stained sections which showed glycogen reduction in the low-dose group, and its accumulation in the high-dose group, compared to the control, respectively. Imunopositivity in hepatocytes for Ki-67 protein, a known marker for proliferation, showed a decrease in low-dose group, while in high- dose group was detected its increase compared to the control, respectively. Stereological analysis confirmed initial histological observation. Caspase 3 immunopositivity, a known marker for apoptosis, proved to be decreased in hepatocytes in both acrylamide-treated groups when compared to the control. One the other hand, immunopositivity was increased in non-parenchymal cell, predominantly in Kupffer cells, in comparison to the control. Immunopositivity for CD68, a marker for Kupffer cells, proved to be decreased in both acrylamide-treated groups when compared to the control. Population of the mast cells, visualized on toluidine blue (TB) stained sections, showed its increase in both acrylamide-treated groups, in comparison to the control. The increase was especially prominent regarding a degranulated subpopulation of these cells. Subsequent stereological analysis confirmed histological findings. Serum analysis showed increased activity of aspartate aminotransferase (AST), and decreased activity of alanine aminotransferase (ALT) in both AA-treated groups, while the activity of alkaline phosphatase (ALP) was increased in low-dose, but decreased in high- dose group compared to the control, respectively. The concentration of total serum proteins as well as concentration of C reactive protein (CRP) did not show any major changes among the experimental groups. Body weight measurements showed that all acrylamide-treated rats lost their body weight as opposed to the control rats whose body mass increased. Present results suggest a prominent hepatotoxic potential of acrylamide which might alter the microstructural features and functional status in hepatocytes of immature liver. Acrylamide may cause significant perturbation in liver functionality which may be reflected from cellular to the tissue level, thereby endangering the whole body’s homeostasis