Variation in FCN1 affects biosynthesis of ficolin-1 and is associated with outcome of systemic inflammation.

Ficolin-1 is a recognition molecule of the lectin complement pathway. The ficolin-1 gene FCN1 is polymorphic, but the functional and clinical consequences are unknown.The concentration of ficolin-1 in plasma and FCN1 polymorphisms in positions -1981 (rs2989727), -791 (rs28909068), -542 (rs10120023), -271 (rs28909976), -144 (rs10117466) and +7918 (rs1071583) were determined in 100 healthy individuals. FCN1 expression by isolated monocytes and granulocytes and ficolin-1 levels in monocyte culture supernatants were assessed in 21 FCN1-genotyped individuals. FCN1 polymorphisms were determined in a cohort of 251 patients with systemic inflammation. High ficolin-1 plasma levels were significantly associated with the minor alleles in position -542 and -144. These alleles were also significantly associated with high FCN1 mRNA expression. The level of ficolin-1 in culture supernatants was significantly higher in individuals homozygous for the minor alleles at positions -542 and -144. Homozygosity for these alleles was significantly associated with fatal outcome in patients with systemic inflammation. None of the other investigated polymorphisms were associated with FCN1 and ficolin-1 expression, concentration or disease outcome. Functional polymorphic sites in the promoter region of FCN1 regulate both the expression and synthesis of ficolin-1 and are associated with outcome in severe inflammation.

A possible role of the dietary fibre product, wheat bran, as a nitrite scavenger.

We found that wheat bran acted as a nitrite scavenger under conditions similar to those that exist in the stomach. Wheat flour and cellulose did not act as nitrite scavengers. Wheat bran, at a concentration equivalent to that in the stomach after ingestion of about two pieces of whole-wheat bread, reduces the nitrite concentration from 20 microM to about 10 microM in 60 min at pH 3.5 and 37 degrees C. At pH 2.5 and 1.5, most of the nitrite had disappeared in 10 min. At pH less than or equal to 2.5 the nitrite scavenging effect of bran was as efficient as that of ascorbic acid. Ferulic acid, a component of bran, reacted rapidly with 20 microM-nitrite both at pH 3.5 and 1.5, whereas phenolic lignin model compounds only reacted at pH 1.5.

Genotoxicity of heterocyclic amines in the Salmonella/hepatocyte system.

The Salmonella/hepatocyte system was employed to determine the mutagenicity in bacteria as well as the DNA damage induced in mouse hepatocytes following exposure to heterocyclic amines. With hepatocytes from C57BL/6N mice, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) showed a clear mutagenic effect in the Salmonella, while weak mutagenic effects were observed with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-6-methyldipyrido[1,2-a:3',2'-b]imidazole (Glu-P-1), and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). All the compounds induced low levels of DNA damage in the hepatocytes. In vivo pretreatment of mice with the potent monooxygenase inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 50 micrograms/kg) clearly increased both the mutagenicity in the bacteria and the DNA damage induced in the hepatocytes in vitro. Glu-P-2 showed the lowest mutagenic effect but induced more DNA damage at low concentrations than the other compounds when TCDD-pretreated hepatocytes were used. These data indicate that the genotoxic potency of Glu-P-2 in the intact hepatocyte differs from that observed in the bacteria. Treatment of hepatocytes with a-naphthoflavone, a selective inhibitor of polycyclic hydrocarbon-inducible cytochrome P-450 form(s), prior to exposure to the heterocyclic amines completely inhibited the mutagenic effect in the bacteria. In vivo administration of all the heterocyclic amines 4 hr prior to isolating the hepatocytes resulted in DNA damage, and this effect was augmented by TCDD pretreatment of mice. Our data suggest that agents modulating the activity and composition of the cytochrome P-450 system may greatly influence both toxicity and carcinogenicity of these heterocyclic amines.

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells.

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene.

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) have previously been shown to induce dose-dependent DNA strand breaks in primary hepatocytes from mice and rats. In an attempt to determine the relationship between the extent of DNA strand breaks and the formation of specific DNA-carcinogen bound adducts in murine liver, the capability of N-OH-AAF and N-OAc-AAF to induce both DNA single strand breaks and adduct formation in in vivo and in primary hepatocytes was measured. N-OH-AAF induced a low level of DNA damage in F344 rats (10 mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment. The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene (Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene (Gua-C8-AF) 34% versus 67% and 3-(guanin-N2-yl)-2-acetylaminofluorene (Gua-N2-AAF) 11% versus 10%, respectively, for rat and mouse liver. An additional unknown adduct (12%) was detected in mouse liver. Dose dependent DNA binding and formation of individual DNA adducts were observed in rat and mouse primary hepatocytes following 1 h exposure to [ring-3H]-N-OH-AAF (0.1-20 microM) and [ring-3H]-N-OAc-AAF (5-20 microM). The patterns of DNA adducts in mouse and rat primary hepatocytes exposed to N-OH-AAF and N-OAc-AF were similar to those obtained in liver following in vivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon (10(-4) M) completely inhibited DNA damage induced by N-OH-AAF in mouse and partially in rat hepatocytes while DNA damage caused by N-OAc-AAF was only partially inhibited by paraoxon (10(-4) M) in both species. Parallel experiments showed that paraoxon, at low concentration (10(-6) M), did not alter either the level of DNA binding or the pattern of adduct formation in rat hepatocytes treated with N-OH-AAF (20 microM). However, at 10(-4) M paraoxon partially blocked DNA binding (60%) and the formation of Gua-C8-AAF (95%) and Gua-N2-AAF (80%) while Gua-C8-AF was increased two-fold. In mouse hepatocytes paraoxon pretreatment (10(-4) M) inhibited the formation of Gua-C8-AF by 70% following exposure to N-OH-AAF (20 microM). Gua-C8-AAF and Gua-N2-AAF were also inhibited but only at 10(-4) M paraoxon.(ABSTRACT TRUNCATED AT 400 WORDS)

The genotoxicity of aromatic amines in primary hepatocytes isolated from C57BL/6 and DBA/2 mice.

The capacity of the chemical carcinogen 2-acetylaminofluorene (AAF) and its derivates to cause DNA damage in primary mouse hepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsive DBA/2 mice was studied using the alkaline elution technique. Low levels of DNA damage were observed after exposure of hepatocytes to either AAF or 2-aminofluorene (AF) (50-100 microM). Quantitation of metabolites produced from AAF in hepatocytes from untreated C57BL/6 and DBA/2 mice using h.p.l.c. showed a similar metabolic profile with respect to C- and N-hydroxylations. After in vivo pretreatment with the potent monooxygenase inducer TCDD (50 micrograms/kg), N-hydroxylation in the C57BL/6- and DBA/2-derived hepatocytes increased 25- and 5-fold, respectively. However, the C-hydroxylation pathways were still responsible for approximately 90% of the metabolism in cells from both strains. This may explain why only a slight increase in the DNA damage was observed in C57BL/6 mouse hepatocytes after incubation with AF or AAF and no increase in DNA damage was seen in the DBA/2 hepatocytes isolated from TCDD treated animals. Both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N- OAc -AAF) caused clear dose-dependent increases in DNA strand breaks (5-100 microM), suggesting that N-hydroxylation was the rate limiting step in the activation process of AAF leading to the DNA damage. Treatment of hepatocytes with paraoxon, an inhibitor of microsomal deacetylase activity, prior to exposure to either N-OH-AAF or N- OAc -AAF completely inhibited the damage caused by N-OH-AAF, while the damage caused by N- OAc -AAF was only partially inhibited. This suggests that these compounds are causing genotoxic effects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene (N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effective genotoxic agent, causing DNA strand breaks at low doses. Depletion of cellular glutathione by pretreatment with diethyl maleate, increased the sensitivity of the cells to the damage induced by N-OH-AF. These data indicate that glutathione may play an important role in the detoxification of N-OH-AF in mouse hepatocytes.