Caratterizzazione del rischio da esposizione a β-esaclorocicloesano (β-HCH) mediante modelli cellulari animali
Author(s)
Rossi, Cristina
Date Issued
June 28, 2016
Type
Doctoral Thesis
Abstract
The β-hexachlorocyclohexane (β-HCH) is a very stable and accumulable isomer of Lindane, a well-known organochlorine pesticide. The HCHs are banned in all developed countries but to date, high concern still exists for the environment, animal and human health due to contaminated sites.
Nowadays, the indirect environmental contamination by OCPs is mainly due to the illegal and/or wrong accumulation of wastes in industrial areas. In 2006, a rural area in Italy devoted to dairy cow farming (the Sacco River Valley, Lazio Region), was found to be contaminated by HCHs: in particular, the β-HCH was found in soil, forages and cow milk as well as in sapwood and bark of poplars growing near the Sacco River. In addition, the β-HCH was found in blood serum of the cows farmed in that area.
The aim of this work was monitoring the effects of the β-HCH on human and animal cell models, to estimating the risk of exposure for both farm animals and operators of the agro-livestock sector.
Two type of immune cells were selected for this study: the bovine Peripheral Blood Mononuclear Cells (PBMCs) and a specific type of immune cells (human T lymphocytes), called Jurkat. The range of β-HCH (from 0.1 to 1000 μM) was chosen on the basis of previous studies and on the basis of β-HCH levels found in vivo on bovine reared in the Sacco River Valley. All the trials were performed incubating PBMCs and Jurkat for 2 and 7 days.
In this study, in vitro tests used to monitoring effects of β-HCH were the following: cell viability test (XTT test); Trypan Blue Exclusion (TBE) assay, for monitoring the number of living cells; Lactate Dehydrogenase (LDH) release assay; Bromodeoxyuridine (BrdU) incorporation assay, for monitoring the proliferation cells activity; Dichorodihydrofluorescein Diacetate (DCHF-DA) assay, for monitoring the intracellular reactive oxygen species concentration; real-time PCR (rt-PCR) for monitoring the mRNA expression of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (Gpx) and Glutathione-S-Transferase (GST)).
The metabolic activity of bovine PBMCs and Jurkat cells increased at 2 days of exposure only in cells exposed to β-HCH 1 μM; instead, after 7 days of exposure, and at higher concentrations of β-HCH (10-100-1000 μM), a decrease of metabolic activity was observed. Results obtained by the DCFH-DA assay showed an increase of Reactive Oxygen Species on bovine PBMCs and Jurkat exposed to β-HCH, both after 2 days of exposure and after 7 days of exposure, also at concentration levels (β-HCH 0.1-1 μM) comparable to those found in vivo in blood serum of cows reared in the Sacco River Valley. Besides, it was observed an increase of activity of detoxification enzymes (SOD, CAT, Gpx, and GST), both on bovine PBMCs and Jurkat cells. The increase of expression of mRNA of SOD, CAT, Gpx and GST was observed only on bovine PBMCs exposed to β-HCH 100 and 1000 μM, after 7 days of exposure.
The LDH assay results suggest that, β-HCH does not clearly affect the integrity of the cell membrane in the range of exposure levels tested.
The BrdU incorporation assays showed rather different results according to the different mitogens and times of exposure; only the highest concentration of β-HCH (1000 μM) induce a decrease of proliferation activity of PBMCs stimulated by PWM and Con A at both incubation time. Instead, PBMCs stimulated with PWM, exhibited a gradual reduction of proliferative activity from 0.1 μM to 100 μM β-HCH, only after 2 days of exposure. A rather different picture has been observed stimulating PBMCs with the mitogen ConA. After 2 days of exposure, no effects have been observed in PBMCs exposed to β-HCH 0.1-100 μM. Instead, after 7 days of incubation, it was seen a decrease of the proliferative capacity of PBMCs exposed to 100 μM β-HCH.
On the basis of these results, we can affirm that, the β-HCH induces, both in cells of the bovine immune system, that in human lymphocyte cells, in vitro, an increase of cellular oxidative stress, even at doses comparable found in vivo in in blood serum of cows reared in the Sacco River Valley. Furthermore, the β-HCH is able to induce a cellular metabolic activity response, and an activation of main detoxifying enzymes, to bring the cell in a state of oxidative homeostasis.
Future objectives are to monitor the presence / absence of markers of lipid peroxidation such as malondialdehyde (MDA), and the mechanism of interaction between the β-HCH, and the immune system cells. Finally, it would be interesting to confirm the hypothesis of activation of cell death pathways for apoptosis.
Additional information
Dottorato di ricerca in Scienze e tecnologie per la gestione forestale e ambientale
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