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Abstract-A method was developed to isolate lipophilic compounds efficiently from small aliquots of blood plasma and test these for total dioxin-like toxic potency using recombinant rat (H4IIE) and mouse (Hepa1c1c7) hepatoma cell lines, containing the firefly (Photinus pyralis) luciferase gene under trans-activational control of the aryl hydrocarbon receptor (AhR). For this experiment, blood plasma was used originating from eider ducks (Somateria mollissima) that had been dosed with 3,3Ј,4,4Ј-tetrachlorobiphenyl (PCB-77) or with the technical PCB-mixture Clophen A50. For each sample the CALUX (chemical-activated luciferase expression) response of both the fat-containing organic extract and the fat-free, cleaned extract were compared with data from chemical analyses of these samples. The CALUX responses for the extracts were converted into so-called CALUX TEQs (TCDD equivalents), using a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard curve. The CALUX TEQs in both fatty and cleaned extracts correlated significantly with PCB-77 or PCB-153 levels (depending on the dosage group) determined in blood plasma using gas chromatography-mass spectrometry (GC-MS). For PCB-77 a toxic equivalency factor (TEF) of 1.5 ϫ 10 Ϫ3 was calculated based on these correlations. In addition, PCB-118 and PCB-156 levels in abdominal fat (assessed with GC with electron capture detection) and hepatic ethoxyresorufin O-deethylase activities correlated well with the CALUX TEQs in both fatty and cleaned blood plasma extracts, suggesting the TEQ levels in blood offer a good measure for internal dose. Plasma cholesterol and triglyceride levels were determined as a measure of lipid content, in 10-l aliquots of blood plasma using enzymatic spectrophotometric determination. In conclusion, we have demonstrated that the CALUX assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) AhR-active compounds in small aliquots of blood plasma. The limit of detection for the CALUX assay is currently less than 0.1 fmol (32 fg) TEQ, which corresponds with the amount of TEQs present in 0.1 to 1 ml of blood plasma in environmentally exposed species or man.

A method was developed to isolate lipophilic compounds efficiently from small aliquots of blood plasma and test these for total dioxin-like toxic potency using recombinant rat (H4IIE) and mouse (Hepa1c1c7) hepatoma cell lines, containing the firefly (Photinus pyralis) luciferase gene under trans-activational control of the aryl hydrocarbon receptor (AhR). For this experiment, blood plasma was used originating from eider ducks (Somateria mollissima) that had been dosed with 3,3Ј,4,4Ј-tetrachlorobiphenyl (PCB-77) or with the technical PCB-mixture Clophen A50. For each sample the CALUX (chemical-activated luciferase expression) response of both the fat-containing organic extract and the fat-free, cleaned extract were compared with data from chemical analyses of these samples. The CALUX responses for the extracts were converted into so-called CALUX TEQs (TCDD equivalents), using a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard curve. The CALUX TEQs in both fatty and cleaned extracts correlated significantly with PCB-77 or PCB-153 levels (depending on the dosage group) determined in blood plasma using gas chromatography-mass spectrometry (GC-MS). For PCB-77 a toxic equivalency factor (TEF) of 1.5 ϫ 10 Ϫ3 was calculated based on these correlations. In addition, PCB-118 and PCB-156 levels in abdominal fat (assessed with GC with electron capture detection) and hepatic ethoxyresorufin O-deethylase activities correlated well with the CALUX TEQs in both fatty and cleaned blood plasma extracts, suggesting the TEQ levels in blood offer a good measure for internal dose. Plasma cholesterol and triglyceride levels were determined as a measure of lipid content, in 10-l aliquots of blood plasma using enzymatic spectrophotometric determination. In conclusion, we have demonstrated that the CALUX assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) AhR-active compounds in small aliquots of blood plasma. The limit of detection for the CALUX assay is currently less than 0.1 fmol (32 fg) TEQ, which corresponds with the amount of TEQs present in 0.1 to 1 ml of blood plasma in environmentally exposed species or man.

Dietary exposure to nitrate and nitrite occurs via three main sources; occurrence in (vegetable) foods, food additives in certain processed foods and contaminants in drinking water. While nitrate can be converted to nitrite in the human body, their risk assessment is usually based on single substance exposure in different regulatory frameworks. Here, we assessed the longterm combined exposure to nitrate and nitrite from food and drinking water. Dutch monitoring data (2012)(2013)(2014)(2015)(2016)(2017)(2018) and EFSA data from 2017 were used for concentration data. These were combined with data from the Dutch food consumption survey (2012)(2013)(2014)(2015)(2016) to assess exposure. A conversion factor (median 0.023; range 0.008-0.07) was used to express the nitrate exposure in nitrite equivalents which was added to the nitrite exposure. The uncertainty around the conversion factor was taken into account by using conversion factors randomly sampled from the abovementioned range. The combined dietary exposure was calculated for the Dutch population (1-79 years) with different exposure scenarios to address regional differences in nitrate and nitrite concentrations in drinking water. All scenarios resulted in a combined exposure above the acceptable daily intake for nitrite ion (70 µg/kg bw), with the mean exposure varying between 95-114 µg nitrite/kg bw/day in the different scenarios. Of all ages, the combined exposure was highest in children aged 1 year with an average of 250 µg nitrite/kg bw/day. Vegetables contributed most to the combined exposure in food in all scenarios, varying from 34%-41%. Food additive use contributed 8%-9% to the exposure and drinking water contributed 3%-19%.Our study is the first to perform a combined dietary exposure assessment of nitrate and nitrite while accounting for the uncertain conversion factor. Such a combined exposure assessment overarching different regulatory frameworks and using different scenarios for drinking water is a better instrument for protecting human health than single substance exposure.