Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethgal concentrations of inorganic arsenic

Inorganic arsenic (jAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress. Glutathione related enzymes including glutathione reductase (GR) and glutathione S-transferase (GST) also play key roles in these processes. In this study the regulatory effects of inorganic arsenite (As111) on the activities of GSH-related enzymes were investigated in cultured human keratinocytes. Substantial increases in GR enzyme activity and mRNA levels were shown in keratinocytes and other human cell lines after exposure to low, subtoxic, micromolar concentrations of As111 for 24 h. Upregulation of GSH synthesis paralleled the upregulation of GR as shown by increases in glutamatecysteine lyase (GeL) enzyme activity and mRNA levels, cystine uptake, and intracellular GSH levels. Glutathione S-transferase activity was also shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells. Overall the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves upregulation of parts, but not all of the GSH system and counteracts the acute toxic effects of jAs. The upregulation of GR has not previously been shown to be an integral part of this response, although GR is critical for maintaining levels of reduced GSH.

Arsenic, mode of action at biologically plausible low doses : what are the implications for low dose cancer risk?

Arsenic is an established human carcinogen. However, there has been much controversy about the shape of the arsenic response curve, particularly at low doses. This controversy has been exacerbated by the fact that the  mechanism(s) of arsenic carcinogenesis are still unclear and because there are few satisfactory animal models for arsenic-induced carcinogenesis. Recent epidemiological studies have shown that the relative risk for cancer among populations exposed to ≤60 ppb As in their drinking water is often lower than the risk for the unexposed control population. We have found that treatment of human keratinocyte and fibroblast cells with 0.1 to 1 μM arsenite (As^III) also produces a low dose protective effect against oxidative stress and DNA damage. This response includes increased transcription, protein levels and enzyme activity of several base excision repair genes, including DNA polymerase β and DNA ligase I. At higher concentrations (> 10 μM), As induces down-regulation of DNA repair, oxidative DNA damage and apoptosis. This low dose adaptive (protective) response by a toxic agent is known as hormesis and is characteristic of many agents that induce oxidative stress. A mechanistic model for arsenic carcinogenesis based on these data would predict that the low dose risk for carcinogenesis should be sub-linear. The threshold dose where toxicity outweighs protection is hard to predict based on in vitro dose response data, but might be estimated if one could determine the form (metabolite) and concentration of arsenic responsible for changes in gene regulation in the target tissues.

The determination of urea in wine – a review

The concentration of urea in wine is not routinely measured in Australian laboratories, but has been examined in studies of yeast metabolism and the formation of ethyl carbamate, a known carcinogen. For alcoholic beverages that may contain high levels of urea, steps have been taken to reduce the concentration of urea and therefore prevent ethyl carbamate production. Methods for the determination of urea in wine can be grouped into three categories that indicate how selectivity for urea is achieved; those based on colour-forming reactions, enzymatic hydrolysis and chromatographic separation. The two dominant methods used by research groups over the past fifteen years for the determination of urea in wine are based on the urea/ammonia test kit available from Boeringer Mannheim/R-Biopharm and the reaction of urea with 1-phenyl-1,2-propanedione-2-oxime; both are time-consuming and labour-intensive, but involve relatively straightforward and well-established procedures. However, other options are available that may be better suited to the desired application and the instrumentation available in any particular laboratory.

Total arsenic accumulation in yabbies (Cherax Destructor Clark) exposed to elevated arsenic levels in Victorian gold mining areas, Australia

Arsenic is a proven carcinogen often found at high concentrations in association with gold and other heavy metals. The freshwater yabby, Cherax destructor Clark (Decapoda, Parastacidae), is a ubiquitous species native to Australia's central and eastern regions, with a growing international commercial market. However, in this region of Australia, yabby farmers often harvest organisms from old mine tailings dams with elevated environmental arsenic levels. Yabbies exposed to elevated environmental arsenic were found to accumulate and store as much as 100 μg/g arsenic in their tissues. The accumulation is proportional to the concentration of arsenic in the sediment and is high enough to be of concern for people who eat the yabbies. A comparison of arsenic levels in wild and lab-fed animals also was performed. Although there was no significant difference in the level of arsenic in the various organs of the wild animals, the animals purchased from a yabby farm showed a significantly higher arsenic concentration in their hepatopancreas (3.7 ± 0.9 μg/g) compared to other organs (0.6–1.8 μg/g). Furthermore, after a 40-d exposure to food containing 200 to 300 μg/g inorganic arsenic, arsenate (As[V])-exposed animals showed a significant increase in tissue-specific arsenic accumulation, whereas arsenite (As[III])-exposed animals showed a lower, nonsignificant increase in As uptake, primarily in the hepatopancreas. These results have important implications for yabby growers and consumers alike.

The influence of specific luminal factors on the colonic epithelium : high-dose butyrate and physical changes suppress early carcinogenic events in rats

INTRODUCTION Although luminal delivery of butyrate is one putative mechanism by which biology of the colonic epithelium might be influenced by changes in luminal contents, there is a paucity of supportive cause–effect evidence. This study aimed to directly establish whether distal colonic butyrate delivery is able to alter the response of the distal colonic epithelium to a carcinogen.
METHODS Groups of male Sprague-Dawley rats with chronically intubated colons received infusions of 80 mM butyrate or 0.9 percent saline into distal colon two or five times daily. Three weeks after exposure to azoxymethane (15 mg/kg subcutaneously), the density of aberrant crypts was quantified in distal colon.
RESULTS Infusions of 0.5 ml twice daily, whether containing saline or butyrate, decreased the number of aberrant crypt foci by 45 percent compared with rats receiving no infusions (P = 0.004, analysis of variance). Similar results were obtained when infusions were restricted to the postinitiation phase. When infusions were increased to 1 ml five times daily, saline infusions similarly suppressed aberrant crypt formation (38 percent), but butyrate infusions suppressed it to a greater degree (by 64 percent; P = 0.02 compared with saline infusion, t-test).
CONCLUSIONS High levels of butyrate delivery to the distal colonic lumen alter the epithelial response to a carcinogen in otherwise healthy rats. This finding directly supports the notion that the effects of butyrate on cells in vitro do occur in vivo provided a sufficient dose is delivered. The effect of infusion of liquid per se on the epithelial response highlights the potential impact physical changes alone can have on the colonic epithelium.

Arsenic speciation in the freshwater crayfish, Cherax destructor Clark

Arsenic is a proven carcinogen that is found in the soil in gold mining regions at concentrations that can be thousands of times greater than gold. During mining arsenic is released into the environment, easily entering surrounding water bodies.
The yabby (Cherax destructor) is a common freshwater crustacean native to Australia's central and eastern regions. Increasing aquaculture and export of these animals has led us to question the effects of mine contamination on the yabbies themselves and to assess any potential risks to consumers. This study determined the species of arsenic present in a number of organs from the yabby. Several arsenic contaminated dam sites in the goldfields of western Victoria were sampled for yabby populations. Yabbies from these sites were collected and analysed for arsenic speciation using high performance liquid chromatography–inductively coupled plasma-mass spectrometry (HPLC–ICP-MS). Results showed that type of exposure influenced which arsenic species was present in each organ, and that as arsenic exposure increased the prevalence of inorganic arsenic species, mostly As(V), within the tissues increased. The bioaccessibility of the arsenic present in the abdominal muscle (the edible portion for humans) of the yabbies was assessed. It was found that the majority of the bioaccessible arsenic was present as inorganic As (III) and As(V).

Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection.

Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.

Exposure to As(III) and As(V) changes the Ca²⁺-activation properties of the two major fibre types from the chelae of the freshwater crustacean Cherax destructor

Arsenic is a known carcinogen found in the soil in gold mining regions at concentrations thousands of times greater than gold. Mining releases arsenic into the environment and surrounding water bodies. The main chemical forms of arsenic found in the environment are inorganic arsenite (As(III)) and arsenate (As(V)). Yabbies (Cherax destructor) accumulate arsenic at levels comparable to those in the sediment of their environment but the effect on their physiological function is not known. The effects of arsenic exposure (10 ppm sodium arsenite, AsNaO2 - 5.7 ppm As(III)) and 10 ppm arsenic acid, Na2HAsO4·7H2O - 2.6 ppm As(V)) for 40 days on the contractile function of the two major fibre types from the chelae were determined. After exposure, individual fibres were isolated from the chela, "skinned" (membrane removed) and attached to the force recording apparatus. Contraction was induced in solutions containing increasing [Ca(2+)] until a maximum Ca(2+)-activation was obtained. Submaximal force responses were plotted as a percentage of the maximum Ca(2+)-activated force. As(V) exposure resulted in lower levels of calcium required for activation than As(III) indicating an increased sensitivity to Ca(2+) after long term exposure to arsenate compared to arsenite. Myosin heavy chain and tropomyosin content in individual fibres was also decreased as a result of arsenic exposure. Single fibres exposed to As(V) produced significantly more force than muscle fibres from control animals. Long-term exposure of yabbies to arsenic alters the contractile function of the two major fibre types in the chelae.