ULTRASTRUCTURAL CHANGES IN THE LIVER OF INTRAVENOUS HEROIN ADDICTS

; e ultrastructural research has a decisive role in gathering the knowledge on the liver's response to the infl uence of some drugs. ; e aim of the study was to perform an ultrastructural analysis of the liver in chronic intravenous heroin addicts. ; e study involved the autopsy conducted on  bodies of intravenous heroin addicts and  control autopsies. ; e liver tissue was fi xed in glutaraldehyde and moulded with epon for investigation purposes of ultrastructural changes. ; e analysis was performed using the method of transmission electron microscopy. In the group of intravenous heroin addicts, the liver autopsy samples showed degenerative vesicular and fat changes, chronic active and persistent hepatitis, cirrhosis, reduction in the amount of glycogen in hepatocytes, as well as the Kupff er cell's dominant hypertrophy. Various changes occur in organelles, plasma membrane of hepatocytes and biliary channels as well as in the nucleus. ; e most important ultrastructural fi ndings include: hyperplasia and hypertrophy of the smooth endoplasmic reticulum, which is histologically proven vesicular degeneration of hepatocyte occurring as a result of the increased synthesis of enzymes of smooth endoplasmic reticulum due to chronic intravenous heroin intake, and the presence of continuous basal membrane followed by transformation of the sinusoids into capillaries (in the cases of chronic active hepatitis and cirrhosis) which leads to a disorder of microcirculation and further progress of cirrhosis.


Introduction
e liver has a key role in removing lipophilic substances from the plasma including both morphine and its derivative heroin.Metabolites of these substances are extracted from the body through enzymes in the hepatocyte.Morphological changes in the liver tissue are accompanied by its changed function which results in diff erent metabolism of heroin and other toxins used simultaneously with heroin (alcohol, medicines).us, the eff ects of their abuse are changed and often surprising (, , , ).
e liver shows a characteristic of adaptation: its function increases under the infl uence of various medicines.The adaptation occurs due to the increased production of enzymes that take part in metabolic destruction.is phenomenon is known as enzyme induction.Such changes can be quantitative and manifested in subcellular damage causing hypertrophy or hyperplasia of hepatocytes known as hepatomegaly ().The electron microscopic (EM) research has the decisive role in gathering the knowledge on the liver's response to the infl uence of some drugs.It is known that most medicines cause biochemical process such as conjugation, hydrolysis and reduction, which are associated with oxidative enzyme system of mixed function (, ).These changes occur in smooth endoplasmic reticulum (SER) ().e discovery of the change in SER under the infl uence of most medicines is expected.e most common response is proliferation of SER, which is proved in patients treated with phenobarbital, difenilhidantion, diazepam and oral contraceptives, but also in chronic heroin addicts.In the case of the induction of proliferation of SER, SER type  component is increased, although views are controversial.Further, common changes are dilatation and vesicular transformation of SER cistern, which was noticed during the treatment with imurran and tetracycline ().ere are some results which suggest that damage of SER by medicines is insignifi cant in the human liver when compared to the animal liver.When human hepatocytes are concerned (contrary to rodents hepatocytes), SER is more abundant then rough endoplasmic reticulum (RER).RER is less studied, and its dilatation and degranulation during treatment with oral contraceptives and fenil-butazon derivates are established.Dilatation, vesiculation and RER extension are found during the treatment with fenacetine and in hypervitaminosis A ().Triguero et al. () performed an analysis of liver biopsies of  intravenous (IV) heroin addicts.At the ultrastructural level, they analyzed  centrolobular sinusoids and compared these results with  sinu-soids from  liver biopsies from controls.EM observation shows the thickening of the sinusoidal wall related to endothelial cell hypertrophy, the increase of the area of the extension of Ito cell and fi brosis of Disse space.Cell hypertrophy can represent hyperactivation of the sinusoid functional capacity of the cells, which initiates fi brogenesis of Disse space. is newly formed mechanical barrier has the power to prevent free exchange of materials through Disse space and can protect the liver from heroin's toxic influence.

Material and Methods
The study includes analysis of the  autopsies,  of which were from the group of IV addicts, and  autopsies served as controls (corpses of young and healthy people who died of mechanical traumas that did not affect the liver).The liver extracts (from  to ) taken during the autopsy were fi xed in glutar-aldehyde and the tissue was moulded in epon for the ultrastructural research.e analysis was performed using the method of transmission electron microscopy JEM  CX JEOL.

Results and Discussion
Various changes occurred in organelles, the plasma membrane of hepatocytes and biliary channels, as well as in the nucleus.

Changes in the cells membranes
The swelling and the thinning of microvilli on the vascular pole accompanied by the protrusion cytoplasm in Disse space were found.This phenomenon is known as shedding figure and represents an unspecific response to shedding (Figure ).Much more important is the widening of intercellular space together with the fearing of desmosomes.

Changes in the nucleus
The condensation of the chromatin and destruction of the nucleus was dominant (Figure ).

Degenerative changes
In the case of serious degeneration type lipid changes, depletion of glycogen in the cytoplasm of hepatocyte was noticeable (Figure ).

Infl ammatory changes
A mesenchymal inflammatory reaction was present in the lobules, portal and periportal areas.Kupffer's and sinusoidal endothelial cells were increased in the lobules (Figure ).In the cytoplasm of Kupffer's cells there were various inclusions, corpuscles, above all, granules of the lipofuscin pigment, hemosiderin, ceroids and biliary inclusions.The expanded Disse space contained the cells of infl ammation of the lymphocyte type and polymorphonuclears which are closely related with hepatocytes.
An important change was capillarization of the sinusoid (Figure ).First, the thin amorphous basal membrane was created along the vascular pole.The basal membrane was followed by the increase of collagen fibres in Disse space, but also in the intercellular space.Those damages caused a disorder of microcirculation and further progress of cirrhosis.

Chronic active hepatitis
In chronic active hepatitis, lymphocyte-plasma cells infi ltrate extended from the portal spaces nearly to the parenchyma (, ).Lymphocyte-plasma cells showed diff erent stages of maturation and were stuck between hepatocytes.During this process, their cell membranes were in close contact.In the case of a limited lysis of the hepatocyte cytoplasmic membrane, the extensions of activated lymphocytes were invaginated into hepatocyte, which is known as emperipolesis (Figure ).Plasma cells were in close connection with the hepatocyte.
ere were phagosomes in macrophages, and together with the fibroblasts they increased protein synthesis thereby enabling the multiplication of collagen.With the multiplication of fi brils, hepatocytes became isolated from the food source which led to irregular lobular organization.Collagen was especially prominent in Disse space near the vascular pole of hepatocyte.e lumen of sinusoids was reduced because of the hypertrophic sinusoidal macrophages (Figure ), and multiplied lymphocytes and plasma cells.Condensation of the collagen fi bres caused intralobular septa which connected the portal zones with the lobular centre damaging lobular structure even more.Besides fi brosis and mononuclear thick infi ltrate, cholangiole were multiplied in the portal space, but had the shape of solid stripes without lumen.

Chronic persistent hepatitis
In port space, there were crowded lymphocytes, histiocytes, plasma cells and fibroblasts, accompanied by proliferation of bile channels.The border plates were intact and the lobular structure was preserved.Hepatocytes were very often without changes, although they sometimes contained HBsAg (the surface antigen of the Hepatitis-B-Virus).In the periphery, the stasis of the bilirubin pigment was found and it was the result of periportal inflammation and fibrosis.The multiplied biliary channels were surrounded by the collagen sheaf, but they had a lumen and basal membrane, as well as microvillus on apical poles.Mitochondria of the hepatocytes changed shape and size due to the paracrystaloid inclusions (Figure ).Sometimes, a nucleoid corpuscle could be seen.

Cirrhosis
In cirrhosis, the nodules in Disse space showed different level of dilatation, as well as increased EM thickness ().The presence of the continuous basal membrane with transformation of the sinus into capillaries was described (Figure ).Bundles of collagen fi bres around the basal membranes were polarized in parallel formations, thus creating thick or thin sheaths.
e forming of basal membranes and sinusoidal capillarization (Figure ) played an important role in the fi brogenesis of Disse spaces.Basal membranes surrounded the portal space and proliferating sinuses, while sinusoidal capillarization represented focus for the occurrence of cirrhotic fi brogenesis.Collagen fi bres were formed even when the basal membrane was not present ().The increase of the reticular and collagen fibres was found in both extended intercellular spaces and cirrhotic nodules which is known as pericellular fi brosis.It has been suggested that a changed Disse space represents a barrier for diffusion of substrates from the sinusoids.That is why the pathological microvillous borders in some occur as an adaptive response.Ito cells of the perisinusoidal spaces had the main role in the synthesis of collagen fi bres.Mitochondrias had greater dimension compared to the megamitochondrias.In hepatocytes, there were Mallory corpuscles.Cholestasis was present in the hepatocytes and small canals and was accompanied by the hypertrophy of Golgi zone and promination of SER ().

Conclusion
e most important ultrastructural fi ndings in the liver of intravenous heroin addicts include: -hyperplasia and hypertrophy of the smooth endoplasmic reticulum which is histologically proven vesicular degeneration of hepatocyte occurring as a result of the increased synthesis of enzymes of SER due to chronic IV heroin intake; -the presence of a continuous basal membrane accompanied by the transformation of sinusoids into capillaries (in cases of chronic active hepatitis and cirrhosis), which causes disorders of microcirculation and further progress of cirrhosis.

SER
e changes in organellesThe ultrastructural changes were the most prominent in the smooth and the rough ER, and less prominent in the mitochondrias, namely prolifera-tion, dilatation and vesicular transformation dominated in SER cisterns, especially in type  (Figure).RER degranulation and fragmentation were also present (Figures , ) that is, separation of ribosomes and their dilatation (Figure ).The mitochondrias changed their size and shape, i.e. they became polymorphous with the thickening of their matrix (Figure ).The swelling of mitochondrias and even the forming of giant mega-mitochondrias were not rare.The criste in them were shortened (Figure .) or fragmented followed by a simultaneous occurrence of crystal inclusion and myelinic figures.The lysosomes were not directly affected by drugs.The changes in them were of secondary nature and are the result of digestion of products of the damaged membranes of other organelles.e autophagal vacuoles, the increased number of lipofuscin pigment granule, and paracrystal inclusions-myelin figures were found in the cytoplasm of hepatocyte, and even in the cytoplasm of biliary epitheliums.(Figure .).