Jorma Mäki-Paakkanen

Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation

The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p<0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p<0.05 and p<0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR.

Association Between the Clastogenic Effect in Peripheral Lymphocytes and Human Exposure to Arsenic Through Drinking Water

We describe the association between structural chromosome aberrations (CAs) and parameters of exposure to arsenic among 42 individuals exposed to arsenic through well waters in Finland. The median concentration of arsenic in the wells was 410 μg/l, the total arsenic concentrations in urine (As‐tot) was 180 μg/l, and in hair 1.3 μg/g, for current users (n = 32) of contaminated wells. Urinary arsenic species and CAs were also analyzed in eight control individuals from the same village who consumed water which contained arsenic <1.0 μg/l (detection limit). Increased arsenic exposure, indicated best by increased concentrations of arsenic species (inorganic arsenic, methylarsonic acid (MMA), dimethylarsinic acid (DMA)) in urine, was associated with increased frequency of CAs. The increased urinary ratio of MMA/As‐tot and the decreased ratio of DMA/As‐tot were associated with increased CAs when all aberration types, including gaps, were considered. Associations between CAs and arsenic exposure indicators were stronger among current users than among persons who had stopped using the contaminated well water for 2–4 months before sampling (ex‐users, n = 10). Furthermore, there was a positive but not statistically significant association between CAs and arsenic in hair among the current users, but not among the ex‐users, who still had relatively high arsenic concentrations in hair. The results suggest that the effect observed in the present study reflects relatively recent arsenic exposure. Environ. Mol. Mutagen. 32: 301–313, 1998

Cytogenetic effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(50H)-furanone (MX) in rat peripheral lymphocytes in vitro and in vivo

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting Salmonella mutagen found in wood pulp chlorination effluents and chlorinated drinking water. In cultured rat peripheral lymphocytes, MX induced significant dose-related increases in sister-chromatid exchanges (SCEs) and chromosome aberrations at doses of 20-60 micrograms/ml and of 60-80 micrograms/ml, respectively. MX produced primarily chromatid-type as opposed to chromosome-type aberrations. The peripheral lymphocytes of male and female rats exposed to MX by gavage 5 days a week for 14-18 weeks showed significant dose-related increases in SCEs at both levels of exposure (30 and 45-75 mg/kg) in both sexes. The present results demonstrate for the first time that MX is genotoxic in vivo.

Toxicological effects of emission particles from fossil- and biodiesel-fueled diesel engine with and without DOC/POC catalytic converter

There is increasing demand for renewable energy and the use of biodiesel in traffic is a major option when implying this increment. We investigated the toxicological activities of particulate emissions from a nonroad diesel engine, operated with conventional diesel fuel (EN590), and two biodiesels: rapeseed methyl ester (RME) and hydrotreated fresh vegetable oil (HVO). The engine was operated with all fuels either with or without catalyst (DOC/POC). The particulate matter (PM1) samples were collected from the dilution tunnel with a high-volume cascade impactor (HVCI). These samples were characterized for ions, elements, and polycyclic aromatic hydrocarbon (PAH) compounds. Mouse RAW264.7 macrophages were exposed to the PM samples for 24 h. Inflammatory mediators, (TNF-α and MIP-2), cytotoxicity, genotoxicity, and oxidative stress (reactive oxygen species [ROS]) were measured. All the samples displayed mostly dose-dependent toxicological activity. EN590 and HVO emission particles had larger inflammatory responses than RME-derived particles. The catalyst somewhat increased the responses per the same mass unit. There were no substantial differences in the cytotoxic responses between the fuels or catalyst use. Genotoxic responses by all the particulate samples were at same level, except weaker for the RME sample with catalyst. Unlike other samples, EN590-derived particles did not significantly increase ROS production. Catalyst increased the oxidative potential of the EN590 and HVO-derived particles, but decreased that with RME. Overall, the use of biodiesel fuels and catalyst decreased the particulate mass emissions compared with the EN590 fuel. Similar studies with different types of diesel engines are needed to assess the potential benefits from biofuel use in engines with modern technologies.

Exposures to drinking water chlorination by-products in a Russian city.

Exposures to water disinfection by-products (DBPs) via ingestion of drinking water, and dermal absorption and inhalation during showering/bathing were assessed in the city of Cherepovets, Russia, which uses heavy chlorination to disinfect organic-rich surface water. Concentrations of DBPs (mean +/- standard deviation) in tap water were the following: total trihalomethanes (THMs) 205 +/- 70 micrograms/l, five haloacetic acids (HAAs) 150 +/- 30 micrograms/l, and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (mutagen X or MX) 160 +/- 50 ng/l. Concentrations of THMs and HAAs exceeded the corresponding US standards by a factor of 2.5, while MX concentrations were the highest ever reported. The mutagenic activity of tap water extracts in the Salmonella TA-100 assay was 14,900 net revertants/l. Concentrations of chloroform in breathing zone air in bathrooms during showering were 330 +/- 260 micrograms/m3, shower room air at an industrial plant 2,600 +/- 1,100 micrograms/m3, and bedrooms of local residents 2 +/- 2 micrograms/m3. The mean concentration of chloroform was 3.2 micrograms/m3 in exhaled air samples collected before showering and 110 micrograms/m3 after showering. Data on water ingestion and water use practices in the general population and for pregnant women were collected using questionnaires and diaries. Due to concerns over microbiological safety of water, average daily consumption of non-boiled tap water in pregnant women was only 0.01 l/day, while consumption of boiled tap water was 0.81 l/day. This resulted in low ingestion exposures to volatile THMs. Inhalation and dermal absorption determined total exposures to these compounds. HAAs and MX persist in boiled water and drinks resulting in high ingestion exposures. Several brands of inexpensive home water filters were tested for removal of these compounds. To demonstrate a method of exposure reduction in a sensitive subpopulation, the most efficient filters were given to a group of pregnant women. These women and a control group of pregnant women without filters maintained water ingestion diaries for two weeks. The use of home filters resulted in reduction of exposures to HAAs by a factor of three and a greater reduction in exposures to MX.

Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems.

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.

Investigation of Co-genotoxic Effects of Radiofrequency Electromagnetic Fields In Vivo

Abstract Verschaeve, L., Heikkinen, P., Verheyen, G., Van Gorp, U., Boonen, F., Vander Plaetse, F., Maes, A., Kumlin, T., Mäki-Paakkanen, J., Puranen, L. and Juutilainen, J. Investigation of Co-genotoxic Effects of Radiofrequency Electromagnetic Fields In Vivo. Radiat. Res. 165, 598–607 (2006). We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 μg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Pulmonary inflammation and tissue damage in the mouse lung after exposure to PM samples from biomass heating appliances of old and modern technologies

Current levels of ambient air fine particulate matter (PM(2.5)) are associated with mortality and morbidity in urban populations worldwide. In residential areas wood combustion is one of the main sources of PM(2.5) emissions, especially during wintertime. However, the adverse health effects of particulate emissions from the modern heating appliances and fuels are poorly known. In this study, health related toxicological properties of PM(1) emissions from five modern and two old technology appliances were examined. The PM(1) samples were collected by using a Dekati® Gravimetric Impactor (DGI). The collected samples were weighed and extracted with methanol for chemical and toxicological analyses. Healthy C57BL/6J mice were intratracheally exposed to a single dose of 1, 3, 10 or 15 mg/kg of the particulate samples for 4, 18 or 24h. Thereafter, the lungs were lavaged and bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation, cytotoxicity and genotoxicity. Lungs of 24h exposed mice were collected for inspection of pulmonary tissue damage. There were substantial differences in the combustion qualities of old and modern technology appliances. Modern technology appliances had the lowest PM(1) (mg/MJ) emissions, but they induced the highest inflammatory, cytotoxic and genotoxic activities. In contrast, old technology appliances had clearly the highest PM(1) (mg/MJ) emissions, but their effect in the mouse lungs were the lowest. Increased inflammatory activity was associated with ash related components of the emissions, whereas high PAH concentrations were correlating with the smallest detected responses, possibly due to their immunosuppressive effect.

Induction of chromosome aberrations by styrene and vinylacetate in cultured human lymphocytes: dependence on erythrocytes

Two vinyl monomers, styrene and vinylacetate, were tested for their ability to induce chromosome aberrations in cultured human lymphocytes. The effects of a 24-h treatment (48 h after culture initiation) were studied both in whole-blood cultures (with 2 X 10(8) erythrocytes/ml) and in isolated lymphocytes (with 4000 erythrocytes/ml). Styrene produced a clear dose-dependent increase in chromatid-type aberrations in whole-blood cultures (0.5-6 mM) and a weaker effect in cultures of isolated lymphocytes (1-4 mM). A statistically significant elevation in aberrations was observed at 2 mM in the former culture type and at 1 mM in the latter. These results support earlier studies on the importance of erythrocytes in the metabolic activation of styrene, but also suggest that a part of this activation occurs in the lymphocytes themselves. Vinylacetate (0.125-2 mM), the more potent clastogen of the two monomers tested, induced a distinct dose-dependent increase in chromatid-type aberrations and a slight elevation in chromosome-type breaks in both culture types. The lowest concentration giving a positive result was 0.25 mM. The clastogenic effects of vinylacetate were somewhat more pronounced in isolated lymphocytes than in whole blood. Vinylacetate is known to be rapidly hydrolyzed in vitro to acetaldehyde, which probably explains the positive result.

Induction of mutation, sister-chromatid exchanges, and chromosome aberrations by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in Chinese hamster ovary cells

The strong bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was tested for the induction of mutation at the Na/K ATPase locus to ouabain resistance (OuaR), sister-chromatid exchanges (SCEs), and chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells without metabolic activation. MX increased the frequency of OuaR mutants in CHO cells when the cells were treated with it in PBS (effective dose range 2-3 micrograms/ml) or in medium (McCoys 5A) without serum (effective dose range 20-30 micrograms/ml). MX also induced SCEs in CHO cells, at 0.19-1.5 microgram/ml, exposure in PBS; at 6-24 micrograms/ml, exposure in medium; and at 3-24 micrograms/ml, exposure in medium plus 2.5% fetal calf serum. The maximum induction of SCEs was about 1.5-2.5-fold compared with control level, irrespective of exposure conditions (PBS, medium or medium plus serum). The most pronounced genotoxic effect of MX was observed in CAs (100% aberrant cells at the dose level of 4 micrograms/ml, exposure in PBS) which were mainly of the chromatid type. In general, MX was more toxic to CHO cells treated in PBS compared with exposure in medium or medium plus 2.5% serum.