Toxicology and Industrial Health

Not only is skin cancer by far the most common human cancer but also the incidence of skin cancer has been increasing at an alarming rate in recent decades. Fortunately, most people now realize that sun exposure causes unattractive photoaging and skin cancer, so they do apply sunscreens conscientiously. However, until recently, most sunscreens did not adequately protect against ultraviolet A (UVA) radiation. Although UVA is indeed less erythrogenic and less carcinogenic than UVB, UVA directly causes photoaging and enhances UVB-induced skin cancer. Furthermore, recent research demonstrates that UVA combined with environmental pollutants (including cigarette smoke) significantly increases the risk of skin cancer. Similarly, previous research demonstrated another synergy between environmental pollutants and UV: When ozone exposure precedes UV exposure, there is enhancement of UV-induced depletion of protective vitamin E from the skin's stratum corneum. This article reviews experimental evidence that environmental pollutants (such as benzo[a]pyrene (BaP), a commonly used index of environmental pollution) are photosensitizers that generate reactive oxygen species (ROS) when exposed to UVA radiation. This in turn causes oxidative and genetic damage, leading to unattractive photodamage and carcinogenesis.

The exposure of the skin to Ultraviolet (UV) radiation is a stressful event and the skin has multiple innate defense mechanisms to counter this threat. For instance, oxidatively damaged nerve cells will express neuroglobin, a hexa-coordinate heme protein, to scavenge free radicals such as nitric oxide (NO). Likewise, keratinocytes will express various anti-oxidant enzymes such as superoxide dismutase (SOD), which will defend the cells against oxidative threats. Nonetheless, cells will still express free radicals during excessive irradiation. A fundamental question that needs to be asked is: what do these cells communicate to one another during such stressful events? This paper will present results of an in vitro study in which dorsal root ganglion were irradiated with UV radiation to elicit oxidative events such as release of NO and calcitonin gene-related peptide (CGRP), a potent neuropeptide. Cell survival was also examined using the standard MTT assay. A plant-based neuroglobin mimic called phytoglobin was used as an NO scavenger to see if control of NO would influence CGRP expression and cell survival. Results of this study demonstrate that control of NO expression in irradiated nerve cells can influence CGRP expression and can also increase cell survival rates. Following this preliminary study, controlled amounts of the nerve cell growth media were added to cultures of normal human keratinocytes and the keratinocytes were allowed to interact with the nerve cell media for 24 h. Following this treatment period, human microarrays were run on the keratinocytes to see what genes were influenced in the keratinocytes as a result of contact with the irradiated nerve cell growth media. It was found that several critical genes expressed by the keratinocytes including NO synthase (NOS1), superoxide dismutase 1 (SOD1), transglutaminase 1 and 3 (TGM1 and TGM3), metallopeptidase inhibitor 1 (TIMP1), and filaggrin (FLG) were clearly influenced by the damaged nerve cells.

Inhaled tobacco smoke comes in direct contact with few organs such as mouth, lungs, and stomach. Cigarette smoke (CS) in lungs has been extensively studied. However, limited data exist on its effect on skin, and there are no long-term experimental studies suggesting toxic effects on skin. Even though it is generally accepted that CS is among the main factors of skin aging, the number of experimental studies showing this aging effect is limited. We hereby studied the effect of long-term exposure to CS on the skin of hairless mice in combination with or without ultraviolet (UV) light. In addition, we investigated potential skin protection by a potent antioxidant namely procyanidine-rich French maritime pine bark extract (PBE) pycnogenol. Male and female hairless SKH-2 mice were exposed for 10 months to tobacco smoke and/or UV light in vivo, and their effects on skin were investigated. Some biophysical parameters such as development of erythema, transepidermal water loss (TEWL), and skin elasticity were measured. The results show that UV and CS may be acting synergistically, as shown by the enhanced TEWL, erythema values, epitheliomas, and squamous cell carcinomas (SCCs) observed, whereas PBE seems to protect skin against SCC.

Beta-carotene has been thought to protect against oxidative stress generated by ultraviolet radiation and thus prevents skin cancer and skin aging (Biesalski and Obermueller-Jevic, 2001). However, nothing is known about its potential effects against other environmental sources of oxidative stress such as ozone (O₃) in skin. Intake of oral β-carotene supplements before exposure to sunlight (and thus inevitably also to O₃) has been recommended on a population-wide basis. However, although some studies have shown β-carotene as providing skin protection as an antioxidant, other studies using skin cells in culture have shown that β-carotene may have unexpected prooxidant properties (Obermüller-Jevic, et al., 2001). Given this, there is an ongoing debate regarding the protective or potentially harmful role(s) of β-carotene in human skin. In this study, the effect of β-carotene on ozone’s effects on the skin of hairless mice was assessed. After feeding a diet supplemented with 0.5% β-carotene for 1 month, mice were subjected to O₃ exposure (0.8 ppm 6 h/day; 7 days) and the induction of proinflammatory markers such as tumor necrosis factor-α (TNFα), macrophage inflammatory protein 2 (MIP2), and inducible nitric oxide synthase (iNOS), and markers of oxidative stress, heme-oxygenase-1 (HO-1), were quantitated. The data showed that β-carotene downregulated the induction of TNFα, MIP2, iNOS, and HO-1 in response to O₃. We conclude that β-carotene provides protection against O₃-induced skin oxidative stress in vivo, which is consistent with a protective role for β-carotene in the skin.

Nerve cells are very responsive to weak pulsed electromagnetic fields (EMFs). Such non-ionizing radiation, with frequencies of 0–300 Hz and 0.1–100 mT, can affect several cellular activities, with unusual dose–response characteristics. The present study examined the effect of a 2-h exposure of synaptosomes on a system generating a peak magnetic field of 2 mT. We evaluated the changes of the synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal Ca²⁺ concentration, and the release of free iron and F2-isoprostanes. O₂ consumption and ATP production remained unchanged in exposed synaptosomes. The intrasynaptosomal Ca²⁺ concentration decreased slowly and no depolarization of the synaptosomal membrane was detected. Finally, the release of free iron and F2-isoprostanes by synaptosomal suspensions also remained unchanged after EMF exposure. These results indicate that the physiological behavior of cortical synaptosomes was unaffected by weak pulsed EMFs.

Genetic modifications caused by chronic exposure to low levels of toxic metals may activate stress-signaling pathways, thus increasing cancer incidence among affected individuals. The aim of this study was to evaluate the relationship between exposure to heavy metals and the incidence of chromosomal aberrations and DNA lesions in a chronically exposed population by using specific biomarkers. The study included 156 subjects divided into two major groups: exposed individuals (in a heavy metal contaminated region, Maramures, Romania) and non-exposed population, as control group (Cluj, Romania). We compared the results of two cytogenetic methods for the detection and quantification of DNA lesions and chromosomal aberrations in normal human cells: Single Cell Gel Electrophoresis or Comet assay and Cytokinesis Block Micronucleus assay. The methods were performed on lymphocytes isolated from whole blood in density gradient. The basal DNA lesions and chromosomal aberrations were evaluated, as well as the repair capacity of the supplementary lesions induced by genotoxic agents such as ionizing radiations. Our results showed a great interindividual variability in the basal level of the DNA lesions and chromosomal aberrations, between and within the groups, the most affected being the heavy metals-exposed groups. Non-exposed subjects from rural area Cluj appeared to be more susceptible to the induction of supplementary DNA lesions and chromosomal aberrations by irradiation. The most efficient repair capacity of the radio-induced DNA lesions was observed in the non-exposed Cluj urban group. Both cytogenetic assays (as tools for detection of DNA lesions and chromosomal aberrations) may be used in human biomonitoring studies as indicators of early biological effects induced by exposure to heavy metals.

Degenerative diseases, immune impairment, and premature ageing commonly affect professional categories exposed to severe environmental and psychological stress. Among these, cosmonauts routinely experience extreme conditions due to microgravity, space radiation, altered oxygen supply, physical and mental fatigue during training, spaceflight, and post-flight. Long route aviation pilots display elevated oncogenic risk, connected with cosmic radiation overexposure, and high mortality rates for cardiovascular causes. Engine drivers, like pilots, are affected by health consequences of psycho-emotional stress, and burnout syndrome. The free radical (FR)/antioxidant (AO) imbalance is a common feature in all these pathological conditions. To assess the effective relevance of oxidative stress, we analyzed blood and urine reliable markers of FR production and AO defenses in 12 Russian cosmonauts, 55 airline pilots, 63 train engine drivers, and 50 age-matched controls by measuring the following: (a) lipophilic/hydrophilic low-molecular weight AO and AO enzyme activities, (b) nitric oxide, superoxide anion, hydroperoxide production, and (c) urinary catecholamine/serotonine metabolites and lipoperoxidation markers. Cosmonauts showed elevated granulocyte superoxide and nitric oxide production, increased erythrocyte superoxide dismutase activity and glutathione oxidation, and drastically decreased plasma/leucocyte lipophilic AO levels (P < 0.001–0.01). Aviation pilots, like train drivers, displayed a mild but constant oxidative stress, more pronounced in intercontinental routes pilots, and consistent with lymphocyte chromosomal alterations, DNA oxidation, and cardiovascular malfunction. Results obtained on these selected professionals operating under wearing conditions offer a solid molecular basis for advising the regular monitoring of clinical biochemistry laboratory markers of AO/FR status, to tailor individually specific AO supplementation and diet regimen, and monitor treatment outcomes.

Phase II enzymes are induced primarily through the common electrophile response element (EpRE) signaling. Studies performed in different cell types and with different inducer appear to indicate variation in the upstream signaling pathways involved in the induction of these phase II genes. Nonetheless, whether variation in signaling among phase II genes in the same cell with the same inducer is unclear. This study is designed to answer this question using human bronchial epithelial cells (HBE1 cells) as a model and screening with a variety of protein kinase inhibitors with varying degrees of specificity. Two electrophiles, 4-hydroxynonenal (HNE) and acrolein, induced the expression of phase II genes (GCLC, GCLM, NQO1, NQO2, HO-1, and GSTM-1). Nrf2 silencing significantly decreased the induction of all of these genes, confirming the involvement of Nrf2-EpRE signaling. ERK and p38MAPK inhibitors had no effect, while a JNK inhibitor abrogated the GCLC and GCLM induction by HNE, but not that by acrolein. Among the PKC inhibitors used, one eliminated gene induction by HNE and acrolein, while two others showed no effects. One PI3K inhibitor decreased the induction of GCLM, NQO1, NQO2 and HO-1, but not GCLC and GST-M1; on the other hand, the inhibitory effects of another PI3K inhibitor on gene induction seems to be gene- and inducer- specific. In conclusion, our data suggest that although phase II genes are coordinately induced through Nrf2-EpRE signaling by electrophiles, the upstream signaling pathways involved are gene- and inducer- specific. It is also suggested that commercial kinase inhibitors may produce non-specific effects on phase II gene expression via mechanisms unrelated to their purported specificity.

There are two families of essential fatty acids that must be obtained from the diet: the ω-6 fatty acids consisting of linoleic and arachidonic acids and the ω-3 fatty acids consisting of linolenic, eicosapentaenoic and docosahexaenoic acids (Prog Chem Fats Other Lipids 1968:9;275–348; Recent Pat Cardiovasc Drug Discov 2007;2:13–21; Mini Rev Med Chem 2008;8:107–115). Vegetables and vegetable oils are sources of linoleic and linolenic acids, and the higher ω-3s are obtained from fish. The estimated ratio of ω-6:ω-3 fatty acids in the typical Western diet is about 20:1, whereas, several lines of evidence indicate that a ration of 1:1 would be optimal. Both series of fatty acids can be oxidatively metabolized to a range of products. The oxidative metabolites of arachidonic acid are all proinflammatory and/or prothrombotic, while the corresponding ω-3 metabolites are anti-inflammatory and/or antithrombotic. The imbalanced consumption of the two families of essential fatty acids contributes to a range of diseases. Greater awareness of this problem is leading to increased use of dietary supplements and new products intended to decrease ω-6 consumption while increasing ω-3 intake.

Olive oil, a typical ingredient of the Mediterranean diet, possesses many beneficial health effects. The biological activities ascribed to olive oil consumption are associated in part to its phenolics constituents, and mainly linked to the direct or indirect antioxidant activity of olive oil phenolics and their metabolites, which are exerted more efficiently in the gastrointestinal (GI) tract, where dietary phenolics are more concentrated when compared to other organs. In this regard, we present a brief overview of the metabolism, biological activities, and anticancer properties of olive oil phenolics in the GI tract.

The antioxidant power of the so-called antioxidants is negligible because their rate constants and concentrations are too small to compete with the specialized defense enzymes, like superoxide dismutase (SOD), for the reactive oxygen species. In this short review, we present a number of experimental data of our group, along with the relevant literature data, to show that in-vivo antioxidants increase the systems reliability in other tacks. For example, butylated hydroxytoluene can prevent production of O₂ ^•– in mitochondria, whereas flavonoids can induce expression of antioxidant enzymes, SOD and catalase. We suggest that the timely introduction of antioxidants can provide the beneficial physiological effects through the prophylactic reliability maintenance against reactive forms of oxygen via the hormonal system.

Bioavailability studies in animals and humans fed with extravirgin olive oil demonstrated that hydroxytyrosol and tyrosol, the major simple phenolic compounds in extravirgin olive oil, are dose-dependently absorbed and excreted. Once absorbed, they undergo extensive metabolism; hydroxytyrosol and tyrosol concentrate mainly in the kidney, where they may exert an important role in the prevention of oxidative stress induced renal dysfunction. In this study we monitored the ability of hydroxytyrosol and tyrosol to protect renal cells (LLC-PK1) following oxidative damage induced by H₂O₂. Oxidative stress was evaluated by monitoring the changes of the membrane lipid fraction. Hydroxytyrosol exerted a significant antioxidant action, inhibiting the production of MDA, fatty acids hydroperoxides and 7-ketocholesterol, major oxidation products of unsaturated fatty acids and cholesterol, and thus protecting the cells from H₂O₂-induced damage. Tyrosol, instead, in this experimental model, did not exert any protective effect.

Oxidative stress produced by the dietary or chemical substrates is one of the major causes of liver cell injury. In this study, we compared the effects of two dietary antioxidants, α-tocopherol (α-T) and β-carotene (β-C) against tert-butyl hydroperxide (tBHP)-induced oxidative stress in human hepatoma HepG2 cells. Cell proliferation, lipid peroxidation (LPO), cellular lactate dehydrogenase (LDH), [³H]-aflatoxin B₁(AFB₁)-DNA adduct formation, and cytochrome P450 2E1 (CYP2E1) expression were determined after antioxidants were added to the tBHP-stressed cells. When compared to an ethanol-based control, all biomarkers for the cell damage were significantly increased by treatments. Treatments of β-C or the combination of two antioxidants at 50 ppm for 48 h enhanced cell proliferation ( P  < 0.05) compared to tBHP control. The antioxidative and cytoprotective actions of α-T and β-C, alone or in combination, were associated with modulation of microsomal CYP2E1 expression, corresponding to the regulation of LPO production ( P  < 0.0001). Our results indicate that α-T and β-C may contribute differently to protection of cellular membrane disruption in CYP2E1-expressing HepG2 cells. Moreover, the combination of α-T and β-C appears to impel the greater protection of pathogenic processes of oxidative stress in liver.

This study was conducted to determine the effect of a natural polyphenolic isoflavone antioxidant (Glabridin) on low-density lipoprotein (LDL) oxidation. Determination of the extent of LDL oxidation was done by measuring the formation of Thiobarbituric acid reactive substances (TBARS). After oral administration of licorice-root ethanol extract to healthy subjects for 6 months, the subjects’ oxidative stress level as well as plasma LDL oxidation reduced by 20%. We concluded that dietary consumption of glabridin protects LDL from oxidation.

Whey proteins (WP) are known to contain more cysteine than casein (CAS), so it is suggested that they should ameliorate the oxidative equilibrium in the organisms. To evaluate the influence of a WP-based diet on liver glutathione (GSH) content, male Sprague-Dawley rats were fed for 3 weeks a balanced liquid diet containing either WP or CAS as main source of protein. Liver GSH content was evaluated at the end of the treatment by high performance liquid chromatography (HPLC), both in basal conditions and after oxidative stress induced by CCl₄ acute intoxication. In basal conditions, WP diet significantly increased hepatic GSH in comparison to CAS diet. After CCl₄ intoxication, hepatic GSH was negligibly increased in CAS group, while its increase was much more marked in WP group, so that the difference between the two diets was significant; this suggests that WP provided rats with better ability to increase their GSH synthesis in case of need.

We studied the anti-arthritic activity of glucomannan (GM) isolated from Candida utilis and of Imunoglukán^®, a beta-(1,3/1,6)-d-glucan (IMG) isolated from Pleurotus ostreatus. Adjuvant arthritis (AA) was induced intradermally by the injection of Mycobacterium butyricum in incomplete Freund’s adjuvant to Lewis rats. Blood for biochemical and immunological analysis was collected on experimental days 1, 14, 21, and 28. A clinical parameter – hind paw volume (HPV) – was also measured. The detection of IL-1 alpha, IL-4, TNF alpha, and MCP-1 was done by immunoflowcytometry. On day 28 – the end of the experiment – we determined spectrophotometrically: the total anti-oxidant status (TAS) of plasma samples along with thiobarbituric acid-reacting substances (TBARS) levels in plasma and we assessed the activity of gamma-glutamyl transferase (GGT) in hind paw joint homogenate. The experiments included healthy animals, arthritic animals without treatment, and arthritic animals with administration of glucomannan (GM-AA) in the oral daily dose of 15 mg/kg b.w. and of IMG (IMG-AA) in the oral daily dose of 2 mg/kg b.w. The progress of AA was manifested by all parameters monitored. Both substances had beneficial effects on HPV, TBARS levels, GGT activity, and TAS levels. For cytokine assessment, only IMG-AA samples were selected, considering the significant HPV improvement accompanied with the observed anti-oxidant action. IMG administration had a positive immunomodulating effect on all cytokine plasma levels measured, changed markedly due to arthritis progression. Thus, IMG may be considered as a candidate for combinatorial therapy of rheumatoid arthritis.

Type 2 diabetes is a heterogeneous disease resulting from insulin resistance and/or from a β-cell secretory defect. Hyperglycemia, which occurs during type 2 diabetes, causes disorders of oxidative–antioxidative balance in the cells, leading to increased free-radical formation. Reduced antioxidant capacity is supposed to be one of the causes of the occurrence of complications in type 2 diabetes. The aim of this study was to evaluate lipoperoxidation and plasma antioxidant status in patients with poorly controlled type 2 diabetes with or without complications. In this study, 15 patients with type 2 diabetes without complications and 11 patients with type 2 diabetes with complications were enrolled. The ‘ferric-reducing ability of plasma’ showed no differences between the two experimental groups. A small, nonsignificant, Superoxide dismutase (SOD) activity reduction was observed in patients with diabetes with complications when compared to those patients with diabetes without complications; on the contrary, we found increased lipoperoxidation in patients with diabetes with complications compared with those patients with diabetes without complications. We also observed a positive correlation between malondialdehyde levels and high density lipoprotein or vitamin E in all analyzed patients with type 2 diabetes. Data obtained from our study show that patients with poorly controlled type 2 diabetes with complications have higher lipoperoxidation than patients with complication-free diabetes, although a residual compensatory response to hyperglycemia-induced oxidative stress occurs.

Arsenic (As) is ubiquitous in soils with an average concentration of 5–6 μg g⁻¹ in uncontaminated soils. Heavy metals (Pb, Cd, Cu, Zn, Cr, Fe and Al) were determined using flame or graphite-furnace atomic absorption. The mean concentrations (mg kg⁻¹) of As (29–500 ppm), Fe (8%), Cu (76 ppm), Pb (52 ppm), Ni (90 ppm), Zn (258 ppm), Mg (1.56%), V (143 ppm), Cr (155 ppm), Cd (<2), Sb (0.29) and Hg (0.54) in fallow land soils are within the normal range. The mean As (470 ppm), Fe (10.57%) and Mg (1.67 ppm). We have evaluated how soil spectral response of pyrite and hematite is distributed in order to obtain the discrimination among these different soil compositions and their distribution along the dump area.

In 2003–2006, the distribution of macronutrients and pollutants of environmental interest was investigated in surficial sediments collected from 10 southern Italy harbors selected in four different regions. About 167 stations were sampled to determine levels of total organic carbon, total nitrogen, total phosphorous, trace elements (Al, Cd, Pb, Ni, Cr, Cu, Zn, Hg, As), short- and long-chain aliphatic hydrocarbons (Hy C > 12 and Hy C < 12), and concentrations of persistent organic pollutants (polychlorinated biphenyls, polycyclic aromatic hydrocarbons [PAHs], p-p-Dichlorodiphenyldichloroethylene (4,4′-DDE), and Hexachlorobenzene (HCB). General relationships between studied variables and harbors systems were explored by multivariate statistical approaches. Results show that wide fluctuations are reported for all variables both among harbors and inside each studied system. Principal components analysis suggests that major significance in explaining total average variability is due to lead, copper, zinc, silts, sands, and PAHs. No significance has been observed when testing nonmetric multidimensional scaling distributions relating with the factor “region,” while performing analyses on factor “main human activity,” a higher significance is observed. These results suggest a strong relationship between the main human use of marine systems and observed pollution levels in sediments.

The aim of the present study was to assess the environmental quality of Orbetello lagoon (Tyrrhenian coast, Italy), using a biomonitoring method based on measuring organochlorinated pollutants in the Anguilla anguilla, chosen as sentinel organism, and remote sensing data obtained by elaboration of Landsat 7 Enhanced Thematic Mapper (ETM) data of 2002. Polychlorinated biphenyls and organochlorine pesticides were determined by gas chromatography/mass spectrometry in muscle of European eels captured in seven sites having different levels of pollution in Orbetello lagoon. Remote sensing data and chemical analysis showed the Albegna river as a probable vehicle of pesticides (hexachlorocyclohexanes).