Anna Rita Volpe

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells.

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.

Renal mechanisms in the cardiovascular effects of chronic exposure to inorganic mercury in rats

Male weanling Wistar rats received 200 micrograms/ml of mercury (Hg), as HgCl2, in drinking water for 180 days. At the end of the treatment, systemic arterial blood pressure was augmented, cardiac inotropism was reduced, and heart rate was unchanged. Light and electron microscopical studies of the kidney showed a mesangial proliferative glomerulonephritis in about 80% of the glomeruli. Tubular cells showed reduction of the acid phosphatase activity, which was related to functional abnormalities of the lysosomes. In the 24 hour urine samples of the Hg exposed rats, there was slight reduction of kallikrein activity, but evident proteinuria was not present in all samples. Plasma renin activity was reduced, that of angiotensin I-converting enzyme was augmented, and plasma aldosterone concentrations were unchanged. Mercury was accumulated mostly in the kidney of the Hg treated animals; and the content of Hg in the heart was higher than in the brain. These data show that chronic exposure to Hg acts on the kidney with complex mechanisms of toxicity; these contribute to modify systemic haemodynamics.

Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium

In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half‐life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium‐calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal. To test this possibility, we evaluated the effect of i.c.v. injection of 88u2003nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre‐treated with: (1) saline (control), (2) L‐arginine (L‐Arg), to increase the availability of substrate for NO biosynthesis, (3) D‐arginine (D‐Arg), (4) 3‐[4‐morpholinyl]‐sydnonimine‐hydrochloride (SIN‐1), an NO donor, or (5) CaCl2, a cofactor of brain calcium‐calmodulin‐dependent cNOSI. In additional experiments, the levels of L‐citrulline (the stable equimolar product derived from enzymatic cleavage of L‐Arg by NO synthase) were determined in the brain of vehicle‐ or cadmium‐treated rats. The pressor response to cadmium reached its nadir at 5u2003min (43±4u2003mmHg) and lasted over 20u2003min in controls. L‐Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium‐treated rats compared with controls. Central injection of NG nitro‐L‐arginine‐methylester (L‐NAME) also reduced the levels of L‐citrulline in the brain. Both the magnitude and duration of the response were attenuated by 1.21 and 2.42u2003μmol SIN‐1 (32±3 and 15±4u2003mmHg, P<0.05), or 1u2003μmol CaCl2 (6±4u2003mmHg, P<0.05). Selectivity of action exerted by SIN‐1 was confirmed by the use of another NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Both L‐Arg and D‐Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L‐Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in ability to attenuate the cadmium‐induced pressure effect, L‐Arg and its isomer exerted differential biochemical changes in brain L‐citrulline, as L‐Arg normalized cadmium‐induced reduction in L‐citrulline levels, whereas i.c.v. D‐Arg did not. We conclude that the pressor effect of i.c.v. cadmium is due, at least in part, to reduced NO formation, consequent to inhibition of brain NO synthase. Accumulation of cadmium in the central nervous system could interfere with central mechanisms (including NO synthase) implicated in the regulation of cardiovascular function.

Renal toxicity and arterial hypertension in rats chronically exposed to vanadate.

The effects of 1, 10, or 40 micrograms/ml of vanadium, given for six or seven months as sodium metavanadate in drinking water on cardiovascular and biochemical variables and the electrolyte metabolism of male Sprague-Dawley rats were investigated. At the end of the exposure period, all animals exposed to vanadate had increased systolic and diastolic blood pressure. This effect was not dose dependent and heart rate and cardiac inotropism were not affected. The role of defective renal function and electrolyte metabolism in such effects was supported, in the rats exposed to 10 and 40 ppm of vanadium, by the following changes: (a) decreased Na, + K(+)-ATPase activity in the distal tubules of nephrons; (b) increased urinary excretion of potassium; (c) increase in plasma renin activity and urinary kallikrein, kininase I, and kininase II activities; (d) increased plasma aldosterone (only in the rats treated with 10 ppm of vanadium). The alterations in the rats exposed to 1 ppm of vanadium were: (a) reduced urinary calcium excretion; (b) reduced urinary kallikrein activity; (c) reduced plasma aldosterone. These results suggest that blood hypertension in rats exposed to vanadate depends on specific mechanisms of renal toxicity related to the levels of exposure.

Mithridates VI Eupator of Pontus and mithridatism

We greatly appreciated the paper by Ring and Gutermuth, ‘100 years of hyposensitization: history of allergen-specific immunotherapy (ASIT)’ (1). Some points referring to Mithridates VI of Pontus deserve, in our opinion, to be further addressed (e.g. ‘... King Mithridates... used increased doses of snake venom to make himself immune against the toxin...’). Mithridates VI (132-63 BC), who ruled the northern part of Anatolia and waged a hard-fought war against the Romans, was interested, like other hellenistic sovereigns, in science, particularly poisons and antidotes (Fig. 1). His celebrated ‘universal antidote’ later came to be known as ‘Mithridatium’ (2). Aulus Gellius (Attic Nights 17.16) states that Mithridates used to mix the blood of Pontic ducks, whose flesh was toxic from their ingestion of plants poisonous to humans, with other substances reputed to ‘expel’ poisons. He also apparently obtained immunity to otherwise fatal doses of arsenic by ingesting tiny amounts over many years. His ‘theriac’ recipe was said to contain more than 50 ingredients, consisting of poison counteracting ‘drugs’ (3). He was known to display his ‘immunity’ to poison plots at banquets, inviting his guests to sprinkle his food and drink with deadly substances. Dio Cassius (Roman History 37.13) reports that Mithridates protected himself by taking his secret ‘antidote’ formula every day. Pliny the Elder (Natural History 25.3) states that Mithridates, through experiments, came up with a daily regimen of taking poison along with ‘remedies’. Appian (Mithridatic Wars 12.16) says that Mithridates accustomed himself to poisons by taking small doses every day. The original formula of the famous mithridatium has not survived, but it is unlikely that a snake venom was employed, as stated by Ring and Gutermuth. In fact, because of the enzymatic/proteic nature of their lethal active principia, reptile venoms taken orally are inactivated by proteolysis in the gut, being able to exert their effects only in the presence of lesions of the inner surface of the first digestive tract. The fact that snake venom could be safely ingested was known in antiquity. For example, the Roman writer Lucan (Bellum Civile/Pharsalia 9.614) stated that ‘snake venoms are dangerous only when mixed to the blood’. The word ‘mithridatism’ is currently used to mean tolerance or unresponsiveness to a toxin, which is acquired by taking gradually larger doses of it. The reported continuous per os assumption of ‘hemetics, antidotes, poisons, remedies and/or (unspecified) drugs’ probably determined Mithridates’ resistance to toxins more by functional and/or metabolic changes than by immune mechanisms. In this regard, gut irritation (impairing the absorption of the poison itself as in the case of chronically taken low doses of arsenic) and/or induction/activation of drug-biotransforming enzymes are likely to have been involved. Such resistance was said to have resulted in an unwonted effect when, in 63 BC, Mithridates tried unsuccessfully to empoison himself to avoid to be captured alive by his enemies. Appian states that Mithridates ‘mixed’ the poison and shared the dose with his two young daughters, then swallowed the rest. The two girls died immediately, but Mithridates only became weak (Fig. 2). The composition of this suicide poison is unknown. The effectiveness of poison in rapidly killing Mithridates’ daughters following oral ingestion suggests a nonproteic nature of the poison itself and, therefore, it is unlikely that Mithridates’ tolerance was based on an immunological mechanism. He had shared the single dose with his two daughters and the remaining amount was sublethal, also due to his tolerance. Figure 1 Portrait of Mithridates VI Eupator by Cristiano M. Ferretti. Mithridates is represented as Hercules wearing a lionskin. The sketch was inspired by the sculpture at Louvre museum (courtesy of the artist). Allergy

Vanadate as factor of cardiovascular regulation by interactions with the catecholamine and nitric oxide systems

The effects of 1 μg/ mL of vanadium, given for 12 mo as sodium metavanadate in drinking water, on cardiovascular and biochemical indices of male rabbits were investigated. At the end of the exposure period, vanadium was more accumulated in bones and kidneys than in spleen and liver; the cardiac ventricles and the aorta contained similar amounts of this element. Blood pressure and heart rate were unchanged in the vanadate-exposed animals since the observed decrease of both cardiac inotropism and stroke volume was counteracted by an increase of peripheral vascular resistance, with reduction of arterial blood flow. The arterial levels of sodium, potassium and aldosterone were unmodified by vanadate which, however, strongly raised those of noradrenaline, adrenaline, L-DOPA, and dopamine. Vanadate caused a marked increase of the activity of monoamine oxidase in renal tubules and liver (probably in relation to the increased plasma catecholamine levels) and a reduction of that of glucose-6-phosphate dehydrogenase in the kidney. There was also evidence that vanadium reduces synthesis and/or release of nitric oxide, the endothelium-derived vasodilating factor, likely through a reduced formation from bradykinin. It was concluded that vanadium may represent an environmental factor of altered cardiovascular homeostasis.

Mithridates VI Eupator, father of the empirical toxicology.

We read with pleasure the article by Gunduz et al., titled “Clinical review of grayanotoxin/mad honey poisoning past and present,” that offers an overview of the toxicological aspects of the “mad honey.” However, from a historical point of view and in a toxicological context, an observation seems mandatory. The king of Pontus (in northeast Anatolia), whose allies used grayanotoxin-containing honey against the troops of Pompey the Great, was Mithridates VI Eupator (132–63 BC), not Mithridates IV Philopator Philadelphus, who died circa 150 BC (more than 80 years before the Roman conquest of Pontus) and brother of the grandfather of Mithridates VI. The mistake probably reflects the fact that in ancient Middle East, many kings bore the name “Mithridates” and should be corrected because Mithridates VI Eupator king of Pontus was one of the pioneers of clinical toxicology. Mithridates VI was the author of a book on roots and plants that at those times were one of the most important sources on venoms, which he used to secretly kill his enemies. The interest of Mithridates VI in empirical clinical toxicology is also demonstrated by the experiments that he carried out on prisoners condemned to capital punishment to test poisons and antidotes. Moreover, as he was afraid to be poisoned, he started to protect himself against poisons by taking progressively increasing sublethal doses. The chemical toxicology of them is unknown but there probably was no additive effect. In this way, Mithridates VI acquired resistance to poisoning possibly by enzymatic activation or metabolic functional changes (mithridatism). This, paradoxically, resulted in an unwanted effect: when he understood that he was falling in his enemies’ hands, he attempted to commit suicide, but because of his acquired tolerance, the amount of poison he swallowed did not kill him despite the fact that he walked around rapidly to hasten its action. Consequently, he looked for death by sword. Besides mithridatism, Mithridates VI gave the name to “mithridatum,” a general antidote whose recipe was found in his cabinet by Roman soldiers and carried to Rome by Pompey. Despite Pliny’s criticism, the “mithridatum”, with some modifications, was given the name “theriaca” and used in medicine until the nineteenth century. The intentional use of mad honey to overwhelm enemy forces is one of the first recorded uses of a biotoxin in warfare and is perfectly correspondent to the personality and to the scientific interests of Mithridates VI Eupator king of Pontus.

Chronic Exposure to Vanadate as Factor of Arterial Hypertension in the Rat: Toxicodynamic Mechanisms

Vanadium (mostly as vanadyl) was found to induce arterial hypertension by acting on peptidergic and catecholaminergic systems as well as on Ca2+ homeostasis in vascular and cardiac myocells.