VETERINARSKI ARHIV 69 (3), 135-148, 1999

ISSN 1331-8055 Published in Croatia

Morphological changes in the organs of gilts induced with low-dose atrazine

Stipica Curic¹*, Tihomira Gojmerac², and Marijan Zuric²

¹Department of General Pathology and Pathological Morphology,
Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

²Croatian Veterinary Institute, Zagreb, Croatia

* Contact address:
Prof. Dr. Stipica Curic,
Department of General Pathology and Pathological Morphology, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Heinzelova 55, Croatia,
Phone: 385 1 23 90 111 Fax: 385 1 214 697

CURIC, S., T. GOJMERAC, M. ZURIC: Morphological changes in the organs of gilts induced with low-dose atrazine. Vet. arhiv 69, 135-148, 1999.

ABSTRACT

In this study we have investigated morphological changes in organs of gilts treated with low doses of atrazine, and s-triazine herbicide. Gilts (cross-bred Swedish and German Landrace) received 2 mg atrazine kg^-1 body mass in feed during 19 days of the oestrus cycle. The final day of treatment corresponded to day -3 of the onset of the next expected oestrus. Macroscopically, in treated gilts we have found cystic ovarian degeneration. Histopathologically, mild parenchymatous degeneration of liver was noted, with mild chronic interstitial hepatitis and stenosis, as well as irregular shape of bile ducts. In kidneys we found sub-acute glomerulitis with atrophy of single glomerules and parenchymatous degeneration with desquamation epithelial cells of some tubules and appearance of proteinaceous casts in their lumen. A cereal degeneration of a small number of muscular fibres was noted in the myocard. Multiple follicular cysts and persisting corpus luteum existed in ovaries, while the uterus was in a resting period (dioestrus). Additionally, lymphoid depletion in lymphoid follicles of lymph nodes and spleen was observed, as well as infiltration of lymphocytes in interstitial tissue of liver and kidneys, together with infiltration of eosinophils in liver and lymph nodes.

Key words: atrazine, intoxication, morphological changes, gilts, pig

Introduction

The s-triazines are among the most widely used herbicides in the world. Atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is a selective herbicide commonly used on many crops for the control of annual grasses and broadleaf weeds in Croatian agriculture. Residues of atrazine are found in some crops (NORRIS and FONG, 1983), in soil (GOH et al., 1993), environmental water (VIDACEK et al., 1994) and drinking water samples (GOJMERAC et al., 1994).

In plant species atrazine inhibits the process of photosynthesis (ESSER et al., 1975), and also RNA, protein and lipid synthesis (ASHTON et al., 1973). However, the effects in mammals are less well investigated.

Atrazine possesses high liposolubility and can enter the bodies of animal through water, feed and feed chain or through grazing. Furthermore, it may induce physiological changes without any visible clinical intoxication.

Residues of atrazine and its metabolites after peroral administration of low doses were found in various tissues of chickens (KHAN and FOSTER, 1976; FOSTER et al., 1979), egg layers (SOVA et al., 1992), turkeys (GOJMERAC and KNIEWALD, 1990), and rats (BAKKE et al., 1972; GOJMERAC and KNIEWALD, 1989).

It is documented that sub-acute doses of atrazine impair renal function and have hepatotoxic effects in rats (SANTA MARIA et al., 1986; 1987), and cause interstitial hepatitis and steatosis, as well as dystrophic changes in renal tubules of egg layers (SOVA et al., 1992). In addition, in the same doses it exerted hepatotoxicity and cystic ovarian degeneration in gilts (GOJMERAC et al., 1995; 1996), and in chronic exposure it leads to ovarian epithelial neoplasms in women (DONNA et al., 1989). Moreover, elevated doses of atrazine produce higher frequencies of embryonal deaths, retardation of yolk sac involution and hypoplasia of splenic corpuscles in chick embryos (GOMERCIC et al., 1995).

In the present study we have investigated morphological changes in the organs of gilts after sub-acute exposure to a low dose of atrazine.

Materials and methods

Chemical

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) of c. 99% purity was obtained from Herbos Chemical Industries (Sisak, Croatia) and purified by recrystallisation from ethenol-water before use.

Animals and treatments

Experiment were performed on twenty (10 experimental and 10 control) gilts (cross-bred Swedish and German Landraces) of c. 6-7 months of age, body mass 80-100 kg, which were farm-bred and kept in the same environmental conditions. The duration of oestrus and length of oestrus cycle were carefully observed for two cycles in each animal prior to the experiment. At the onset of oestrus (day 0), manifested by certain behavioural signs, such as "standing to be mounted" the experimental gilts were given 2 mg atrazine kg^-1 body mass in the feed daily for 19 days of the oestrus cycle. The final treatment day corresponded to day -3 of the next expected oestrus (day 0). Animals (control and experimental group) were sacrificed by bleeding according to standard slaughterhouse procedures, 9 days after cessation; that is, 28 days after the beginning of treatment.

Gross pathology and histopathology

All twenty gilts were processed in the same way immediately after death. The abdominal and thoracal wall, as well as the skull was incised, macroscopical pathology was evaluated and samples of heart, lung, liver, spleen, kidney, adrenal gland, thyreoid gland, ovary, uterus, brain, hypophysis cerebri and prescapular and mesenteric lymph nodes were taken. Tissue samples were immediately immersed in 10% formalin at room temperature and fixed for at least 48 h before processing. Fixed specimens were dehydrated through graded alcohols and embedded in paraffin wax; 6 µm thick serial sections were cut, stained with haematoxylin and eosin (H/E), and examined with a light microscope. At least two specimens of each organ were examined from test and control gilts.

Bacteriology and parasitology

Bacteriological examination of test and control gilts was performed on the lung, liver and small and large intestines, and parasitological examination of all small and large intestines, according to established procedures.

Results

No adverse clinical reactions associated with the treatment were recorded.

Macroscopically, visible multiple cysts, approximately 1-2 cm in diameter, were found on both ovaries of each treated gilt. On other examined organs we were not able to macroscopically establish any changes in the tested and control animals.

In the non-treated gilts, no histopathological changes were found in the examined samples.

A mild centrolobular parenchymatous degeneration of hepatocytes (Fig. 1), accompanied by a mild chronic interstitial hepatitis (Fig. 2), was observed in the liver of atrazine-treated pigs. The hepatitis was associated with mononuclear lymphocyte and eosinophil infiltration of interstitial connective tissue. Narrowing and irregular forms of bile canaliculi were also seen (Fig. 2).

Fig. 1. Mild centrolobular degeneration of hepatocytes of atrazine-treated gilt. H/E; 10×3.75; bar=50 µm

Fig. 2. Mild chronic interstitial hepatitis with narrowing and irregular forms bile canaliculi of atrazine-treated gilt. H/E; 10×3.75; bar=50 µm

Histopathological examination of kidneys revealed subacute glomerulitis with atrophy of single glomerulus and mononuclear cell infiltration in the interstitium (Fig. 3). Parenchymatous degeneration and desquamation of epithelial cells of a single proximal convoluted tubules (Fig. 4) were, in some instances, followed by the appearance of proteinaceous casts in the lumen of some convoluted or collected tubules (Fig. 5).

Fig. 3. Subacute glomerulitis with atrophy of glomerules and mononuclear cell infiltration in the interstitium of kidney of atrazine-treated gilt. H/E; 20×3.75; bar=30 µm

Fig. 4. Parenchymatous degeneration and desquamation of epithelial cells of proximal tubules of atrazine-treated gilt. H/E; 40×3.75; bar=20 µm

Fig. 5. Proteinaceous casts in the lumen of tubules of atrazine-treated gilt. H/E; 20×3.75; bar=30 µm

Cereal degeneration of a small number of muscular fibres was noted in the myocardial tissue (Fig. 6).

Fig. 6. Cereal degeneration of a small number of muscular fibres in the myocard of atrazine-treated gilt. H/E; 10×3.75; bar=50 µm

Multiple ovarian follicular cysts (small or large) in different stages of development or regression and cystic degeneration of secondary follicles (Fig. 7) up to the appearance of corpus albicans were observed in both ovaries. A small number of atretic follicles, as well as normal primary or secondary follicles and persisting corpus luteum (Fig. 8) were also seen.

Fig. 7. Multiple ovarian follicular cysts and cystic degeneration of secondary follicle of atrazine-treated gilt. H/E; 20×3.75; bar=30 µm

Fig. 8. Persisting corpus luteum of atrazine-treated gilt. H/E; 20×3.75; bar=30 µm

The uterus was in a state of uterine rest (diestrus) in atrazine-treated animals (Fig. 9).

Fig. 9. The uterus in a state of diestrus of atrazine-treated gilt. H/E; 10×3.75 bar=50 µm

Lymphoid depletion in the lymphoid follicles of prescapular and mesenteric lymph nodes was accompanied by infiltration of eosinophilic granulocytes (Fig. 10).

Fig 10. Lymphoid depletion in the lymphoid follicles of lymph node of atrazine-treated gilt. H/E; 20×3.75; bar=30 µm

Depletion of lymphoid cells was also established in the lymphatic tissue (lymph nodes) of white pulp of spleen.

No histopathological changes were established in the lung, adrenal gland, thyreoid gland, brain or hypophysis cerebri.

Presence of pathogenic bacteria and parasites in the examined organs of the experimental and control animals was not determined.

Discussion

It has been well documented that acute or subacute clinically manifested pesticide intoxication in animals commonly appears with general dysfunction of various organs and tissues, especially those of the digestive and nervous systems. Furthermore, chronic intoxication is usually characterised through general symptoms (debilitation, anorexia, decrease in production and others), but in some cases no symptoms can be observed.

Research regarding clinical symptoms caused with triazine herbicides is very scanty. Peracute or acute intoxication with high doses of poison is well documented in a cattle (JOHNSON et al., 1972; ZDELAR et al., 1972), sheep and horses (EGYED and SHLOSBERG, 1977). In our study we found no clinical symptoms in gilts during the course of observations (19 days). This could be due to low doses of toxin (2 mg kg^-1 body mass) in the feed.

The possible mechanisms of toxic effect and associated lesions in organs and tissues due to herbicide of the triazines group are poorly investigated, especially in swine. In fact, experimental intoxication is most often carried out on laboratory animals in sub-lethal doses and amounts, which are found as a residue in various biological environments, as well in the food of people and animals.

Macroscopically, the only lesions detected in gilts treated with atrazine were multiple cysts, 1-2 cm in diameter, on both ovaries. Histopathologically, diagnosis of multiple follicular cysts is confirmed, in various phases of development and regression. Furthermore, a lesser number of normal primary or secondary follicles or corpus luteum persistent, and a few atretic follicles were also detected. In previous studies (KNIEWALD et al., 1987, 1991) it was demonstrated that atrazine interferes with biochemical pathways in organs with a neuroendocrine function, and with an influence on the gonadotrophic mechanism in rats. Cystic degeneration of ovaries in gilts treated with atrazine is a consequence of a significantly lower level of serum 17 b-oestradiol and significantly higher level of serum progesterone (GOJMERAC et al., 1996). That is the reason why the following oestrus and ovulation are absent, so that non-ruptured follicles become cystic. Corpus luteum persistent and the uterus in a phase of diestrus are a consequence of inadequate serum level of 17 b-oestradiol (GUTHRIE et al., 1972; VAN DE WIEL et al., 1981; GOJMERAC et al., 1996) or increased oestrus cycle caused by atrazine (SIMIC et al., 1994). Detected histopathological changes in organs of gilts could increase the risk of carcinogenesis and subsequentially ovarial epithelial neoplasms (DONNA et al., 1989).

In swine, liver lesions of a non-infectious cause are frequently induced by alimentary substances, such as phytotoxins (MACHIN et al., 1975), mycotoxins (NEWBERNE, 1973) or insecticides and herbicides (ROGERS et al., 1973; MACHIN et al., 1975). Studies regarding the possible hepatotoxic effect of atrazine in pig industry are still lacking.

Histopathological changes in hepatocytes are dependent on doses and duration of intoxication with atrazine. In cases of peracute intoxication with high dosages, diffuse parenchymatous degeneration of hepatocytes is noted (ZDELAR et al., 1972). In cases of subacute intoxication with lesser doses fatty infiltration and degeneration of hepatocytes is observed (SOVA et al., 1992), as well as decrease of glycogen and increase of lipids in liver (MANCIULEA et al., 1980). In cases of chronic intoxication with low doses, fatty infiltration and degeneration of hepatocytes, especially pronounced centrolobulary is described (DSHUROV, 1979). Mild centrolobular parenchymatous degeneration of hepatocytes found in our study could have been induced by proliferation and degeneration of the smooth endoplasmic reticulum caused by the toxic effect of atrazine (SANTA MARIA et al., 1987). Such a hepatotoxic effect of atrazine is manifested through inhibition of key enzymes of gluconeogenesis in liver (SREBOCAN et al., 1975)., as well as decrease in the serum glucose level, and an increase in total serum lipids and activity of serum alanine aminotransferase and alkaline phosphatase (SANTA MARIA et al., 1987). Moreover, subacute intoxication of gilts with atrazine caused parenchymatous degeneration of hepatocytes with a subsequential proliferation of interstitial connective tissues with the appearance of mild chronic interstitial hepatitis, which is subsequent in many cases of intoxication (SCHEUER, 1977). Stenosis and irregularly shaped bile ducts are a consequence of cholestasis, caused by degeneration of the microvilli of epithelial cells of bile ducts, which is caused by the toxic effect of alkaline phosphatase in animals intoxicated with atrazine (SANTA MARIA et al., 1987). Furthermore, in animals treated with atrazine an increase in the activity of serum Y-glutamyltransferase was noted (GOJMERAC et al., 1995), which is an indicator of cholestasis (RICO et al., 1977; ZEPEITZ et al., 1981).

Nephrotoxicity of atrazine is a consequence of its elimination through the kidneys (BAKKE et al., 1972), which leads to an increase in sodium, potassium and chloride in urine, as well as a decrease in creatinine clearance and proteinuria (SANTA MARIA et al., 1986). These pathophysiological changes are a consequence of decreased glomerular filtration which, in our investigations, could have developed due to subacute glomerulitis and atrophy of glomeruli, or decrease of renal tubule reabsorption due to parenchymatous degeneration of tubular epithelial cells and their desquamation with the appearance of proteinaceous debris in lumen of tubule. That is in accordance with other studies in various animal species (ZDELAR, 1972; DSHUROV, 1979; SOVA et al., 1992).

Cereal degeneration of a small number of muscular fibres arose from the toxogenic effects of atrazine (VAN VLEET and FERRANS, 1986).

Cellular infiltrate of lymphocytes in interstitial liver and kidney tissue, as well as eosinophils in liver and prescapular and mesenteric lymph nodes, and lymphoid depletion in lymphoid follicles of lymph nodes and spleen, are subsequent to immunopathological events arising because of the effects of the toxin (VALLI, 1993).

Daily doses of atrazine used in this study (2 mg kg^-1 body mass or 100 mg atrazine kg^-1 diet for 19 days), which represent doses used for agricultural purposes, probably are not accumulated in the animal organism. Nevertheless, because of the obvious appearance of this herbicide in the environment, and on basis of our results, it is clear that subacute poisoning of gilts with low doses of atrazine has a toxic effect on ovaries, liver, kidneys, myocard and lymphatic organs. Thus, the presence of atrazine in the environment, its possible ingestion by food and water, as well as accidental intoxication, could represent a risk for the human population.

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Received: 20 May 1999
Accepted: 16 June 1999

CURIC, S., T. GOJMERAC, M. ZURIC: Morfoloske promjene u organima nazimica izazvane niskim dozama atrazina. Vet. arhiv 69, 135-148, 1999.

SAZETAK

Istrazene su morfoloske promjene u organima nazimica iz intenzivnog uzgoja tretiranih niskim dozama atrazina, s-triazinskog herbicida. Nazimice (krizanci svedskog i njemackog landrasa) dobivale su 2 mg atrazina na 1 kg tjelesne mase u hrani dnevno, tijekom 19 dana estrusnog ciklusa. Posljednji dan davanja odgovarao je s danom -3 od pocetka slijedeceg ocekivanog estrusa. Makroskopski, u tretiranih nazimica nadena je jedino cisticna degeneracija ovarija. Histopatoloski, uocena je blaga parenhimska centrolobularna degeneracija jetre s blagim kronicnim intersticijskim hepatitisom te suzenje i nepravilni oblici zucnih vodova. U bubrezima je naden subakutni glomerulitis, atrofija pojedinih glomerula i parenhimska degeneracija s deskvamacijom epitelnih stanica nekih tubula i pojavom proteinskih odljevaka u njihovom lumenu. U srcanom misicu je nadena vostana degeneracija pojedinacnih misicnih vlakana. U jajnicima su postojale multiple folikularne ciste i perzistentna zuta tijela, dok je maternica bila u fazi mirovanja (diestrus). Takoder, nadena je limfoidna deplecija u limfoidnim folikulima limfnih cvorova i slezene, kao i infiltracija limfocita u intersticiju jetre i bubrega i eozinofilnih granulocita u jetri i limfnim cvorovima.

Kljucne rijeci: atrazin, otrovanje, morfoloske promjene, nazimice, svinja