Isabel Pizarro

SEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules

Most of cisplatin’s citotoxic properties are due to the interaction of the drug with DNA. However, other biological molecules present in the cell cytosol, such as MT (metallothionein) and GSH (glutathione), are potential targets for cisplatin and have been related to its side-effects or with the cellular resistance mechanisms to the drug. Experiments simulating physiological conditions have been performed to study the specific cisplatin metabolites which interact with GSH and MT and to characterize the different drug–biomolecule adducts over time. A combination of size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) techniques has been used to provide sensible multi-elemental detection and structural information of the species of interest. Time dependent transformation of 10 μM cisplatin at neutral pH (7.4) produces different concentrations of the mono-aquo and oligomeric derivatives, as could be confirmed by ESI-MS. No di-aquo derivative was seen to be produced under these conditions at any of the incubation times used. Cisplatin and the oligomeric derivative were incubated with GSH and MT at different drug:biomolecule ratios. Adducts from cisplatin–GSH (1:500) and from cisplatin–MT (1:10) incubations were characterized by SEC-ICP-MS. While both GSH and MT reacted with cisplatin producing different compounds, only GSH reacted with the oligomeric derivative of cisplatin. SEC-ICP-MS experiments showed that, under neutral pH conditions, Cd atoms remained bound to the cisplatin:MT adducts, but Zn atoms were lost. Results were compared with those obtained by in vitro and in vivo experiments with rat kidney, liver and inner ear cytosols.

An approach to the arsenic status in cardiovascular tissues of patients with coronary heart disease

Among non-cancer effects of arsenic, cardiovascular diseases have been well documented; however, few are known about the arsenic fate in cardiovascular tissues. We studied the analytic bioinorganic arsenic behaviour in cardiovascular tissues from an arsenic exposure coronary heart disease patient group from Antofagasta-Chile against a small unexposed arsenic coronary heart patient group. Total arsenic concentrations were measured in pieces of cardiovascular tissues of the arsenic-exposed and unexposed coronary heart patient groups by hydride generation atomic absorption spectrometry (HG-AAS); speciation analysis was made by high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Pieces of auricle (AU), mammary artery (MAM), saphenous vein (SAP) and fat residuals (FAT) were considered in this study. The arsenic concentrations in AU and MAM tissues were significantly different between both groups of patients. Also, it was demonstrated that the AU is an ‘As3+ target tissue.’ Otherwise, linking of the total concentrations of arsenic with conditional variables and variables related to medical geology factors allowed us to infer that the latter are more important for the cardiovascular risk of arsenic exposure in the Antofagasta region. Knowledge of total arsenic and the prevalence of the trivalent ion (As3+) in the AU of patients could contribute to understanding the effect of arsenic on cardiovascular diseases.

Evaluation of arsenic species–protein binding in cardiovascular tissues by bidimensional chromatography with ICP-MS detection

Intracellular As–protein binding in cytosol extracts of auricle and saphenous tissues of As impacted people was evaluated by bidimensional chromatography (size exclusion and FPLC-UV-ICP-MS). The fractionation of cytosol using Superdex (0.1–7 kDa), Phenomenex (1–300 kDa) and MonoQ HR 5/5 columns shows that As is distributed in a wide range of contiguous fractions for each column, the percentages of As in the fractions collected being 8, 25 and 50%, respectively. The FPLC-UV-ICP-MS chromatograms of the fractions from the above columns show that As is bound to biocompounds of different molecular mass through vicinal sulfur groups. The monitoring of S, Cu and P present in the fractions show that As and Cu are usually bound to the same type of proteins, some of which contain P. No significant differences were observed between the auricle and saphenous tissues in the As fractionation behaviour of the cytosol. From the Superdex column it can be deduced that the molecular weight of the peptides associated to As are within the range 330–4600 Da, which could correspond to metallothioneins, glutathione (As(SG)3) and cysteine (As(Cys)3) complexes. The As–proteins of molecular weight within the range 0.3–12.4 kDa from the Phenomenex fractioning could correspond to transferrine and hemoglobin. This work can be considered as a first step in the investigation of As bioaccumulation and the biochemical response of cardiovascular tissues proceeding from individuals chronically impacted by inorganic As when the source for human As uptake is drinking water.

Bioaccessibility and arsenic speciation in carrots, beets and quinoa from a contaminated area of Chile.

Consumption of vegetables grown in arsenic (As)-contaminated soils is an important exposure route to the element for humans. The present study is focused on locally-grown, frequently-consumed vegetables, such as carrots (Daucus carota), beets (Beta vulgaris) and quinoa (Chenopodium) from the As-polluted Chiu Chiu area in Northern Chile. The latter region is affected both by As discharge from copper mining activity and natural As contamination, leading to a high As content in local food and water. For the selected vegetables, the following aspects were investigated: i) Their total As, Cu, Pb, Cr, Cd and Mn content; ii) Arsenic speciation in the edible part of the vegetables by liquid chromatography inductively-coupled plasma mass spectrometry (LC-ICPMS) analysis; iii) Arsenic bioaccessibility in the vegetables during in vitro gastrointestinal digestion; iv) Arsenic species present in the extracts obtained from in vitro gastrointestinal digestion; and v) Arsenic dietary exposure estimates for the assessment of the risk posed by the vegetables consumption. A significant degree of As contamination was found in the vegetables under study, their metal content having been compared with that of similar Spanish uncontaminated products. In vitro gastrointestinal digestion of the studied vegetables led to quantitative extraction of As from carrots and beets, whereas efficiency was about 40% for quinoa. For carrots, only As(III) and As(V) species were found, being their concentration levels similar. In the case of quinoa, around 85% of the element was present as As(V). For beets, inorganic As(V) and unknown overlapped As species (probably arsenosugars) were found. No significant transformation of the original As species was observed during in vitro gastrointestinal digestion. Arsenic dietary exposure values obtained for the three vegetables (0.017-0.021μg As person(-1)day(-1)) were much lower than the JFCFAs safety limit of 50μg As person(-1)day(-1). Therefore, no toxicological risk would be expected from the intake of these vegetables.

Presencia de altos niveles de arsénico en tejidos cardiovasculares de pacientes de áreas contaminadas en Chile

En la Region de Anto-fagasta – Chile la contaminacion cronica por arsenico es causa de numerosas patologias donde se destacan las en-fermedades cardiovasculares. El presente estudio detalla la concentracion de arsenico total y sus principales espe-cies encontradas en tejidos cardiovasculares de pacientes que han requerido cirugia cardiaca. Se relacionan los niveles de As con hallazgos histologicos.

ARSENIC SPECIES-BINDING PROTEINS IN HUMAN CARDIOVASCULAR AND MUSCLE TISSUES

SUMMARY The intracellular As-protein binding in cytosol and methanol–water extract of the auricle and saphene tissues of As impacted people was evaluated by bidimensional size exclusion FPLC-UV-ICP-MS. The fractionation of cytosol using Superdex, Phenomenex and MonoQ HR 5/5 columns, shows that As was distributed in a wide range of contiguous fractions of each column, being 8, 25, 50 % the percentages of As in the collected fractions, respectively. Arsenic a sulphur coelute when FPLC–UV–ICP–MS was applied, which could implicate that As is bound to bio-compounds of different molecular mass through vicinal sulphur groups. The monitoring of S, Cu and P. In the methanol: water extracts a similar study than performed with the cytosol using preparative gel chromatography on Sephadex G-75 and Shephadex G-100 columns. A very low As and protein contain were found in the different fractions of both SEC fractionating series. A similar As–protein association to that found in the cytosol after fractionating with MonoQ HR 5/5 was observed for auricle and saphene. Inorganic and methylated As speciation in the 20 - 26 cytosol fractions obtained within the Phenomenex column was performed by HPLC–ICP–MS using the Hamilton PRP-X100 column. Only As(III) and As(V) were present and the results obtained shows that the As(III)/As(V) ratio is constant in most cases. Direct evidence of the existence of As–binding peptides in auricle and saphene vein from arsenic impacted human beings has have been obtained which was previously reported by means of novo peptide synthesis.

TOTAL AND MAIN As SPECIES PRESENT IN CARDIOVASCULAR TISSUES OF PEOPLE LIVINGIN As CONTAMINATED AREAS

ABSTRACT The concentration levels of As in the Chilean II Region of Antofagasta produces non cancer health outcomes such as cardiovascular diseases and in last term heart attack. On this study, the determination of total As content and main inorganic and organoarsenic species found in three heart tissues (auricle, mammary artery and fat) and the saphene vein of people living in the Chilean II Region, suffering coronary thrombosis has been carried out. Comparison with similar tissues of patients from other non-contaminated areas has been undertaken.The extraction of As species occurred in methanol: water (1:1) and As species determination have been used the tandem HPLC-ICP-MS using the Hamilton PRP X100 anion column. For total As determination has been performed by HG-AAS and ICP-MS.The auricle and in less extend the saphene support the higher As concentration (mean values of 7.7 and 2.5 µg g -1 , respectively), being As(III) the predominant species. Methylarsonate (MA) and dimethylarsinate (DMA) is not a favoured mechanism. The presence of high total As and high As(III) species content in the auricle and saphene of more contaminated people, the damage found in the saphene tissue and the global characteristics of the people under study in which the As stigmas are present in all of them, suggests that As could be involved in the cardiovascular diseases.

Urinary excretion of platinum, arsenic and selenium of cancer patients from the Antofagasta region in Chile treated with platinum-based drugs

BackgroundArsenic exposure increases the risk of non-cancerous and cancerous diseases. In the Antofagasta region in Chile, an established relationship exists between arsenic exposure and the risk of cancer of the bladder, lung and skin. Platinum-based drugs are first-line treatments, and many works recognise selenium as a cancer-fighting nutrient. We characterised the short-term urinary excretion amounts of arsenic, selenium and platinum in 24-h urine samples from patients with lung cancer and those with cancer other than lung treated with cisplatin or/and carboplatin. As - Se - Pt inter-element relationships were also investigated.ResultsThe amounts of platinum excreted in urine were not significantly different between patients with lung cancer and those with other cancers treated with cisplatin, despite the significant variation in platinum amounts supplied from platinum-based drugs. In general, the analytical amounts of excreted selenium were greater than those for arsenic, which could imply that platinum favours the excretion of selenium. For other types of cancers treated with drugs without platinum, excretion of selenium was also greater than that of arsenic, suggesting an antagonist selenium-anti-cancer drug relationship.ConclusionsRegards the baseline status of patients, the analytical amounts of excreted Se is greater than those for As, particularly, for cisplatin chemotherapy. This finding could imply that for over the As displacement Pt favours the excretion of Se. The analytical amounts of excreted Se were greater than those for As, either with and without Pt-containing drugs, suggesting an antagonist Se-anti-cancer drug relationship. However, it seemed that differences existed between As - Se - Pt inter-element associations in patients treated for lung cancer in comparison with those treated for cancer other than lung. Therefore, knowledge obtained in this work, can contribute to understanding the arsenic cancer mechanism and the As - Se - Pt inter-element association for lung cancer and other types of cancer, which in some cases respond at a linear mathematical model.

Identificación y cuantificación del arsénico unido a las proteínas de tejidos cardiovasculares de pacientes sometidos a cirugía de revascularización coronaria

Los efectos de la intoxicacion con Arsenico (As) como enfermedades cardiovasculares (CV), pigmentaciones y oclusiones arteriales coronarias estan asociados con la ingestion de As inorganico a tra-ves del agua de bebida y a exposiciones ambientales. La union del As (III) a proteinas y la metilacion del As podria ser una primera etapa en el mecanismo de detoxificacion.

Magnesium, zinc, arsenic, selenium and platinum urinary excretion from cancer patients of Antofagasta region, Chile: multi-metal approach

Objectives To evaluate the short-term 24 h urinary excretion of platinum, arsenic, selenium, magnesium and zinc in patients with lung cancer and with cancer other than lungs treated with cisplatin or/and carboplatin from Antofagasta, Chile. Design Urine measurements of Pt and Se were made by inductively coupled plasma optical emission spectrometry, As by hydride-generation atomic absorption spectrometry and Mg and Zn by means of flame furnace atomic absorption spectrometry. Setting All samples were provided by the Oncological Centre of Antofagasta Regional Hospital (Region of Antofagasta, Chile). Participants Ninety 24-h urine samples from cancer patients after the infusion of Pt-base drugs and 10 24-h urine samples from cancer patients not treated with metal-base drugs. Main outcome measures Concentrations of Pt, Se, As, Zn and Mg coming from 24-h urine samples. Results Pt excreted was not significantly different between patients with lung and other cancers treated with cisplatin. The excretion of Mg, Zn and Se was greater than As. Then, Pt favours the excretion of essential elements. For lung and other types of cancers treated with drugs without Pt, excretion of Mg, Zn and Se was also greater than that of As, suggesting antagonism Mg-Zn-Se–anti-cancer drug relationship. Conclusions The amounts of Mg, Zn and Se excreted were greater than for As either with or without Pt-containing drugs, suggesting antagonist Mg-Zn-Se–anti-cancer drug relationships. The excretion of As, Mg, Zn and Se is induced by Pt. Knowledge obtained can contribute to understanding the arsenic cancer mechanism and the As-Mg-Zn-Se-Pt inter-element association for lung cancer and other types of cancer.