Maria de Lourdes Pereira

Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: Evaluation by the comet assay

In the present study, the chemoprotective effects of quercetin, rutin and ursolic acid on tert-butyl hydroperoxide (t-BHP)-induced DNA damage in a human hepatoma cell line (HepG2) were investigated by the comet assay. To determine whether protection was due to direct chemical interactions alone or to cellular-mediated responses three different types of treatments were used: simultaneous incubation of cells with individual test compounds and the toxicant; pre-treatment with test compound before addition of the toxicant followed or not by a recovery period. The expression of Hsp70 was quantified by Western blotting to test the involvement of heat shock proteins in the cellular responses to the test compounds. In addition, effects on proliferation were evaluated by the MTT assay. The results show that quercetin and ursolic acid prevented DNA damage and had antiproliferative properties in HepG2 cells suggesting an anticarcinogenic potential for these compounds. The protective effects of quercetin against t-BHP-induced DNA damage seem to be due to both direct effects on t-BHP toxicity and to cellularly mediated indirect effects which reflect the potentiation of the cellular antioxidant defenses. Ursolic acid seems to exert effects only through cellularly mediated mechanisms since it was not protective in simultaneous incubation. Quercetin and ursolic acid also showed to increase the rate of DNA repair. Rutin did not have effects at any level. These results, obtained with liver cells, emphasize and confirm the chemopreventive potential of quercetin and ursolic acid, which may help explain the lower cancer incidence in human population with high dietary intakes of fruits and vegetables. These results also demonstrate that Hsp70 is not involved in the observed effects in HepG2.

Heavy Metals and Human Health

Metals occur naturally in the earths crust, and their contents in the environment can vary between different regions resulting in spatial variations of background concentrations. The distribution of metals in the environment is governed by the properties of the metal and influences of environmental factors (Khlifi & Hamza-Chaffai, 2010). Of the 92 naturally occurring elements, approximately 30 metals and metalloids are potentially toxic to humans, Be, B, Li, Al, Ti, V, Cr, Mn, Co, Ni, Cu, As, Se, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Cs, Ba, W, Pt, Au, Hg, Pb, and Bi. Heavy metals is the generic term for metallic elements having an atomic weight higher than 40.04 (the atomic mass of Ca) (Ming-Ho, 2005). Heavy metals enter the environment by natural and anthropogenic means. Such sources include: natural weathering of the earth’s crust, mining, soil erosion, industrial discharge, urban runoff, sewage effluents, pest or disease control agents applied to plants, air pollution fallout, and a number of others (Ming-Ho, 2005). Although some individuals are primarily exposed to these contaminants in the workplace, for most people the main route of exposure to these toxic elements is through the diet (food and water). The contamination chain of heavy metals almost always follows a cyclic order: industry, atmosphere, soil, water, foods and human. Although toxicity and the resulting threat to human health of any contaminant are, of course, a function of concentration, it is well-known that chronic exposure to heavy metals and metalloids at relatively low levels can cause adverse effects (Agency for Toxic Substance and Disease Registry [ATSDR], 2003a, 2003b, 2007, 2008; Castro-Gonzalez & Mendez-Armenta, 2008). Therefore, there has been increasing concern, mainly in the developed world, about exposures, intakes and absorption of heavy metals by humans. Populations are increasingly demanding a cleaner environment in general, and reductions in the amounts of contaminants reaching people as a result of increasing human activities. A practical implication of this trend, in the developed countries, has been the imposition of new and more restrictive regulations (European Commission, 2006; Figueroa, 2008).

Adverse effects of cadmium exposure on mouse sperm

The effects of cadmium chloride exposure on sperm functional parameters were evaluated on eight-week-old ICR-CD1 male mice administered with a single s.c. injection of 1, 2 and 3 mg CdCl(2)/kg bw. Groups of animals treated with each dose, as well as their respective controls, were sacrificed after 24h to detect short-term (acute) effects and after 35 days. Sperm cells were collected from the epididymis and several parameters of sperm quality and function were evaluated, namely density, morphology, motility, viability, mitochondrial function, acrosome integrity, together with DNA fragmentation assessed by the TUNEL assay. The short-term effects of cadmium chloride resulted in an increased fraction of sperm with abnormal morphology, premature acrosome reaction and reduced motility. Late term effects (after 35 days) included a drastic reduction of sperm cell numbers and sperm motility. An increase in DNA fragmentation was also detected.

Evaluation of nickel toxicity on liver, spleen, and kidney of mice after administration of high-dose metal ion

The toxic effects caused by nickel (Ni) per si were explored by performing in vivo studies on mice following subcutaneous administration of a metallic solution of nickel at 1, 2, 3, and 4 weeks to evaluate the side effects of this metal ion when released from stainless steel implants. Other groups were similarly injected with HBSS and used as controls. Accumulation of Ni ions on liver, spleen, and kidney was assessed by an electrochemical method, adsorptive stripping voltammetry (AdSV) using microelectrodes, and atomic absorption spectrometry (AAS). Alterations of those organs induced by Ni ions were studied, showing that several histological changes had been induced. Chemical analysis and histological features indicate that Ni is partially accumulated in the study organs.

Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond

In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabiting effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it becomes of paramount importance to assess the damages induced by local contamination on these aquatic vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumulation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic damage on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn, Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioaccumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal blood cells might be compromised.

Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice.

The objective of the present work was to evaluate the toxic effects of sodium arsenite, As(III), in mice and the protective effect of 2 antioxidants, hesperidin and lipoic acid, against the observed As(III)-induced toxicity. In each study, mice were assigned to 1 of 4 groups: control, antioxidant, antioxidant + arsenite, and arsenite. Animals were first injected with the vehicle or 25 mg antioxidant/kg BW. After 30 minutes they received an injection of 10 mg arsenite/kg BW or 0.9% NaCl. Two hours after the first injection, the liver, kidney, and testis were collected for histological evaluation. Liver samples were also taken for quantification of arsenic. In mice exposed only to As(III), various histopathological effects were observed in the liver, kidneys, and testes. In mice pretreated with either hesperidin or lipoic acid, a reduction of histopathologic effects on the liver and kidneys was observed. No protective effects were observed in the testes for either of the 2 studied antioxidants. In conclusion, hesperidin and lipoic acid provided protective effects against As(III)-induced acute toxicity in the liver and kidneys of mice. These compounds may potentially play an important role in the protection of populations chronically exposed to arsenic.

Testicular mitochondrial alterations in untreated streptozotocin-induced diabetic rats

Diabetes-induced complications are associated with mitochondrial dysfunction and increasing evidence suggests that diabetes has an adverse effect on male reproductive function. The STZ-induced diabetic rat was used as an animal model for the type 1 form of the disease with the aim of determining its effects in spermatogenesis and testicular mitochondrial function. Several aspects of mitochondrial function were measured, including respiratory and electric potential function, as well as mitochondrial calcium loading capacity. Additionally oxidative stress production, antioxidant levels and possible apoptotic alterations were also evaluated. We observed that diabetic animals present alterations in spermatogenesis in both the testis and epidydimus. However, and surprisingly, the overall results in mitochondrial parameters failed to reveal severe testicular mitochondrial dysfunction in diabetic animals, with the exception of a decrease in calcium load. Taken together, results suggest that in animal models that mimic untreated type 1 diabetes the severe effects of the condition on spermatogenesis are not directly mitochondrial-mediated.

Chromium accumulation and ultrastructural changes in the mouse liver caused by stainless steel corrosion products

Stainless steel (SS) corrosion products were obtained by electrochemical dissolution of SS type AISI 316L. Mice were injected subcutaneously with 0.5 ml of SS solution (containing 283 μg Fe, 69.3 μg Cr and 57 μg Ni) each 72 h, for 10 days or 14 days. After the treatment time, livers were removed and were analysed for: (a) liver wet weight; (b) contents in Fe, Cr and Ni; (c) histological and ultrastructural alterations. Results showed that the percentage of liver weight per animal body weight was significantly higher (p < 0.05) in SS-injected animals than in the control animals. The atomic absorption spectrometry analysis of dry livers showed that chromium, but not iron or nickel, had a significant increase (p < 0.05) in SS-treated mice compared to the control animals. No histopathological differences between 10 and 14 days of SS-injection could be detected, however, massive hepatic degeneration was observed in both groups when compared to the control. These histological changes in SS-treated mice were confirmed at the ultrastructural level, as hepatocytes exhibited an augmentation of vacuoles in their cytoplasm. These actual liver morphological alterations suggest that the hepatocyte function may be hampered, which constitutes a matter of some concern since liver is a blood filtering organ.

Histological effects of iron accumulation on mice liver and spleen after administration of a metallic solution

Special attention has been focused on the toxicity of some metallic species released from implanted materials, which accumulate in vital organs over long periods of time. A set of experiments with mice was designed to investigate the individual effects caused by iron in the liver and spleen. Histological features of these organs were evaluated and slight morphological changes were observed during the treatment time suggesting a negative correlation with the duration of the iron treatment. In addition, to associate the histological changes in the organs with iron accumulation an electrochemical method, adsorptive stripping voltammetry, was chosen to quantify the iron levels in these mentioned organs. The accuracy of the proposed method was checked by atomic absorption spectrometry. Both organs showed elevated concentrations of iron, nearly twofold, 7 days after iron administration compared to control organs.

Impairment of mice spermatogenesis by sodium arsenite

In order to assess the effect of arsenic on the male reproductive impairment in mice, 7-week-old animals were exposed to 7.5 mg sodium arsenite (NaAsO2)/kg body weight, during 35 days (one spermatogenic cycle). One group of animals was sacrificed after exposure, while another group received distilled water for an additional period of 35 days, in order to study the spermatoxic effect and the recovery of spermatogenesis. In mice sacrificed after NaAsO2 exposure, a decrease in testis/body weight ratio and reduction of tubular diameter were observed. Both groups of NaAsO2-exposed animals showed remarkable histopathological changes, such as sloughing of immature germ cells. Animals sacrificed after NaAsO2 exposure showed decreased sperm motility, increased abnormal sperm morphology and decreased sperm viability. The effects of NaAsO2 on sperm motility recovered to normal values after one spermatogenic cycle, while increased sperm abnormality was maintained. However, at this period, a decrease in acrosome integrity was detected. Concerning oxidative stress parameters, animals sacrificed after NaAsO2 exposure showed a decreased selenium-dependent glutathione peroxidase activity, which was not detected after the recovery. Conversely, at this period, total glutathione peroxidase activity increased in exposed animals. These results demonstrate the toxic effects of NaAsO2 on mice spermatogenesis and show the lack of recovery after one spermatogenic cycle.