Ultrastructural Alterations of Peripheral Blood Lymphocytes of White Rats (Rattus norvegicus) Exposed to Lead

MATERIALS AND METHODS

Experimental Animals
A total of 20 male Wistar albino rats (Rattus norvegicus) of the same age weighing 110-130 g, of the King Saud University colony, were used.

Experimental Design
Animals were randomly divided into two groups of 10 rats each and maintained under the standard laboratory conditions including ambient temperature (23-24°C). The animals receieved water and food ad libitum. Following a period of acclimatization (7 days), lead acetate trihydrate was administered via drinking water at the concentration of 0.25% for the exposed group, while sodium acetate replaced lead acetate for the control group. The experimentation period extended for six months. At the end of this period animals were sacrificed and blood was collected from all experimental animals into tubes containing a proper anti-coagulant (ethylenediamine tetracetic acid, EDTA).

Electron Microscopy
The anti-coagulated blood samples were mixed with equal volume of 3% glutaraldehyde in sodium cacodylate buffer (pH 7.2) and kept for 6 h at 4°C. The blood samples were then centrifuged at 3000 rpm for 20 min to separate buffy coats which were then placed in the same fixative for additional 4 h. The buffy coats were subsequently dehydrated through ascending grades of ethanol, cleared in propylene oxide and finally embedded in epoxy resin (Epon: Araldite mixture). Ultrathin sections from buffy coats (70-80 nm) were made on an ultra-microtome (Leica UCT, Austria) using a diamond knife, mounted on copper grids and contrasted with uranyl acetate and lead citrate. The contrasted ultrathin sections were observed under a transmission electron microscope (JEOL, TEM 1011) operating at 80 kV.

RESULTS

Lymphocytes of the Control Animals
Most of peripheral blood lymphocytes of the control animals are the small-sized ones which are round cells having a narrow rim of cytoplasm surrounding a non-lobulated nucleus (Fig. 1). The cytoplasmic contents are scarce and largely involve free ribosomes and tiny rudimentary Golgi complex. There are few dense azurophil granules (lysosomes) and clear vacuoles, but there are no specific granules. Enoplasmic Reticulum (ER) in not well-developed and only few profiles of RER, scattered polyribosomes and reiltively large number of free ribosomes are recognizable. Mitochondria are very few in number. The round nucleus of lymphocytes is often slightly indented, nuclear chromatin is packed at the nuclear periphery and nucleoli are not present (Fig. 2). Plasma membrane has a few pseudopodia. Fewer number of medium-sized lymphocytes are also seen and these cells are identified by their relatively larger nuclei and the more abundant cytoplasmic mass.

Lymphocytes of the Exposed Animals
Lymphocytes in peripheral blood of the exposed animals were the predominant leucocytes, beside neutrophils, observed in the examined ultr-αthin sections. Many lymphocytes revealed nuclear abnormalities, the most common was the various degrees of invagination of the nuclear envelope. The nuclear invagination, in considerable number of lymphocytes, appeared as macroclefts which were deep enough to nearly divide the altered nuclei into two or more portions (Fig. 3 and 4). The resultant nuclear segments were connected by a narrow bridge (s) of chromatin. Widening of the space between the outer and inner nuclear membranes was frequent in the cleaved nuclei showing deep invaginations.

Fig. 1:	Lymphocyte of a control rat showing a round nucleus (N) surrounded by a narrow rim of cytoplasm. Scale bar = 1 μm

Fig. 2:	Lymphocyte of a control rat showing slight nuclear indentation (arrow) and peripheral clumping of the nuclear chromatin (CH). Scale bar = 1 μm

Fig. 3:	Lymphocyte of an exposed rat showing deep invagination (*) of the nucleus (N) which is nearly divided into two portions. Mitochondria (M) are swollen and have deteriorated cristae. Scale bar = 1 μm

Fig. 4:	Lymphocyte of an exposed rat showing segmentation of the nucleus (N) due to several deep invaginations. Mitochondria (M) are swollen. Scale bar = 1 μm

Fig. 5:	Complete separation of the nuclear portions (n) resulting from deep invaginations in a lymphocyte nucleus of an exposed rat. Note the collection of the azurophil granules (*). Scale bar = 1 μm

Mitochondria of the altered cells were swollen and had disoriented or deteriorated cristae. Cytoplasm of these cells contained varied sized vesicles and increased number of azurophil granules. Some lymphocytes showed obvious separation of the nuclear portions resulting from the deep invaginations (macroclefts) which completely cleaved the altered nuclei (Fig. 5). The separated segments were of irregular size and shape and some had obviously condensed chromatin (Fig. 6). Another form of nuclear abnormalities was the marked irregularity of the nuclear envelope which gave the appearance of nuclear protrusions (Fig. 7). Occasionally, the irregular nuclei showed intranuclear cytoplasmic invaginations which were represented by moderately dense bodies delimited by a single membrane and surrounded by a narrow empty space (Fig. 8). These bodies probably represented intranuclear pocket-like structures or pseudo-inclusions. Chromatin condensation was also a form of nuclear abnormalities, the highly condensed chromatin was the heterochromatin with less contribution for euchromatin (Fig. 9). Markedly shrunken nuclei with highly condensed chromatin were seen in some lymphocytes (Fig. 10). Noticeably proliferated rough endoplasmic reticulum was recognized in some lymphocytes which had shrunken nuclei (Fig. 11), as well as in lymphocytes with nearly regular nuclei (Fig. 12). The fine granular content in cisternae of the proliferated RER was revealed on higher magnification of the alterted lymphocytes (Fig. 13).

Fig. 6:	A nuclear portion (*) in a lymphocyte of an exposed rat. Note the marked condensation of the nuclear chromatin, and the large azurophil granule (arrow). Scale bar = 1 μm

Fig. 7:	Nucleus (N) of a lymphocyte of an exposed rat showing obvious protrusions of the nuclear envelope. Note the site of the deteriorated organelles (*). Scale bar = 1 μm

Fig. 8:	Irregular nucleus (N) showing protrusions and moderately dense intranuclear body (arrow) which possibly represents a process of cytoplasmic intranuclear invagination. Scale bar = 1 μm

Fig. 9:	Nucleus (N) of a lymphocyte of an exposed rat showing highly condensed chromatin (CH). Scale bar = 1 μm

Fig. 10:	Lymphocytes of an exposed rat showing obviously shrunken nuclei (N) which have markedly condensed chromatin. Mitochondria (M) are deteriorated. Scale bar = 1 μm

Fig. 11:	Lymphocyte of an exposed rat showing extensively proliferated RER (*). Note the markedly shrunken nucleus (N) which possesses condensed chromatin. Scale bar = 1 μm

Fig. 12:	Lymphocyte of an exposed rat showing proliferated and dilated RER (*). Nucleus (N) is relatively compressed. Scale bar = 1 μm

Fig. 13:	Higher magnification of a lymphocyte of an exposed rat showing markedly dilated RER (*) which contains fine granular material. Nucleus (N) is shrunken and mitochondria (M) are deteriorated. Scale bar = 1 μm

DISCUSSION

Most of the peripheral blood lymphocytes examined in the present study were the small-sized ones. Although the peripheral blood lymphocytes have uniform appearance, but they involve different populations (Bloom and Fawcett, 1975). The majority of these cells are recirculating long-lived ones which participate in cell-mediated immunity. The noticed small-sized lymphocytes has a little cytoplasmic mass, in this respect the activity state of lymphocytes affects the amount of cytoplasm, being larger in medmium- and large-sized lymphocytes (Wheater et al., 1987).

In a previous study (Taib et al., 2004). Comparable mitochondrial changes to that observed here, have been described in hepatocytes of lead-intoxicated rats. Mitochondria is one of the main target organelles in lead toxicity (Aldridge, 1970; Racker, 1970). The present results support the conclusion that mitochondria are highly susceptible to lead toxicity. The observed mitochondrial swelling is probably ascribed to osmolarity changes, which eventually lead to influx of ions and subsequently fluid accumulation with ultimate mitochondrial swelling (King et al., 1983). Lead may also interact with mitochondrial membranes and thus results in impaired electron transport which accentuates the mitochondrial swelling. Mitochondrial swelling has been also described in tissues such as heart, cerebellum and kidney of lead exposed animals (Bull, 1980; Jarrar, 2001). Mitochondria with noticeable membrane changes exhibit compromised oxidative phosphorylation and reduced ATP production, since these processes are largely dependant on the integrity of mitochondrial cristae (Kendall and Scanlon, 1985; Oskarsson et al., 1992).

Presently, considerable number of lymphocytes revealing marked RER proliferation and dilatation were detected in the lead intoxicated animals. It has been stated that lymphocytes with extensive endoplasmic reticulum are cells concerned with protein synthesis (Bloom and Fawcett, 1975). However, these cells which possessed proliferating RER are presumably highly dividing cells, since chronic lead toxicosis has been found to induce lymphocytosis (Basaran and Undeger, 2000). Many peripheral lymphocytes of the currently intoxicated rats disclosed remarkable nuclear chromatin condensation, which has been described as an early morphological apoptotic change (Cohen and Duke, 1984; English et al., 1989). However, to confirm the ability of lead to provoke apoptotic changes in the circulating lymphocytes, further studies at the molecular level should be conducted.

The increased number of azurophil granules, possibly primary lysosomes, in the currently altered lymphocytes is presumably an indication for enhanced autophagic activity related to the presence of degenerated or deteriorated mitochondria. In this case, autophagic activity is considered a removal mechanism of the defective organelles in the affected cells.

In general, the interpretation of lead-induced toxicity of lymphoid cells is based on the fact that lead is a sulfhydryl alkylating agent with a high affinity for subcellular sulfhydryl group (Goyer, 1991). Thus, lead is able to associate with the membrane and intracellular thiols which are important in lymphocyte activation, proliferation and differentiation.

Lymphocytes are known to play a central role in all immunological defensive mechanisms (Bloom and Fawcett, 1975; Wheater et al., 1987). The presently demonstrated ultrastructural changes of lymphocytes of exposed rats undoubtedly affect this role and thus the immune status can be significantly compromised. In this respect, lead is one of the heavy metals that exert immunotoxic effects and it has the ability to impair both cell-mediated immunity and antibody-mediated host resistance (Dean and Murray, 1991). It has been found that chronic lead exposure suppresses cell-mediated immunity parameters including delayed type hypersensitivity and lymphoproliferation in response to mitogens (Faith et al., 1979; Blakely and Archer, 1982). The lead-induced immunosuppression would increase the host susceptibility to pathogens.

Conclusively, the present results indicate that chronic lead exposure can induce subcellular changes in peripheral blood lymphocytes of the exposed animals. Further studies should focus on the mechanism (s) through which lead could provoke such changes using the recent molecular tools.

ACKNOWLEDGMENT

The present research was supported by the Research Center, College of Science, King Saud University (Research Project No. Zoo/ 2005/ 15). The authors gratefully acknowledge this support.