Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040–589?nmol?g^?1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10–10?000 pmol N-(2-hydroxethyl)valine?g^?1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring.

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7-992 nmol g-1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2-250 pmol g-1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040-589 nmol g^-1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10-10 000 pmol N-(2-hydroxethyl)valine g^-1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring

Abstract

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7–992 nmol g^?1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2–250 pmol g^?1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.     

Genotoxic effects of ethylene oxide,propylene oxide and epichlorohydrin in humans: update review (1990-2001)

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.     

Genotoxic effects of ethylene oxide and propylene oxide: a comparative study

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5–10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.     

Mutagenic activity of ethylene oxide and propylene oxide under XPG proficient and deficient conditions in relation to N-7-(2-hydroxyalkyl)guanine levels in Drosophila

Ethylene oxide (EO) and propylene oxide (PO) are direct acting mutagens with high Swain-Scott s-values, which indicate that they react preferentially with ring nitrogens in the DNA. We have previously described that in the X-linked recessive lethal (RL) assay in Drosophila postmeiotic male germ cells EO is, per unit exposure dose, 5-10 times more mutagenic than PO. Furthermore, at the higher dose range of EO tested, 62.5-1000 ppm, up to 20-fold enhanced mutation rates were measured in the absence of maternal nucleotide excision repair (NER) compared to repair proficient conditions. The lower dose range of EO tested, 2-7.8 ppm, still produced a small increased mutation rate but without a significant elevated effect when the NER system is being suppressed. The lowest dose of PO tested, 15.6 ppm, produced only in NER- condition an increased mutation rate. The aim of the present study was to compare the mutagenic effect of EO and PO in the RL assay under XPG proficient and deficient conditions with the formation of N-7-(2-hydroxyethyl)guanine (7-HEG) and N-7-(2-hydroxypropyl)guanine (7-HPG), respectively, the major DNA adducts formed. The formation of 7-HEG and 7-HPG was investigated in Drosophila males exposed to EO and PO as a measure of internal dose for exposures ranging from 2 to 1000 or 2000 ppm, respectively, for 24h. Analysis of 7-HEG and 7-HPG, using a highly sensitive 32P-postlabelling assay, showed a linear increase of adduct levels over the entire dose range. The non-linear dose-response relationship for mutations could therefore not be explained by a reduced inhalation or increased detoxification at higher exposure levels. In analogy with the four times higher reactivity of EO the level of N-7-guanine alkylation per ppm was for EO 3.5-fold higher than that for PO. Per unit N-7-guanine alkylation EO was found to be slightly more mutagenic than PO, whereas PO was the more potent clastogenic agent. While this research has not identified the DNA lesions that cause the increase in repair deficient flies, it supports the hypothesis that efficient error-free repair of some N-alkylation products can explain why these agents tend to be weakly genotoxic or even inactive in repair-competent (premeiotic) germ cells of the mouse and the Drosophila fly.     

Genotoxic effects of inhaled ethylene oxide, propylene oxide and butylene oxide on germ cells: sensitivity of genetic endpoints in relation to dose and repair status

We report here results on forward mutation induction (recessive lethal mutations, RL) in Drosophila spermatozoa and spermatids by the three 1,2-alkyl-epoxides ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO), at doses ranging from 47 to 24,000 ppm h for EO, 375 to 48,000 ppm h for PO, and 24,000 to 91,200 ppm h for BO. The results indicate for EO mutation induction at doses 500-fold below the LD₅₀. In crosses of mutagenized NER⁺ males with NER⁺ females, the 500-fold increase in EO dose from 47 ppm h to 24,000 ppm h resulted in no more than a 17-fold enhanced mutant frequency in spermatozoa. This flat dose–response relationship is primarily the result of efficient repair of EO-induced DNA adducts in the fertilized egg, as was evident from the up to 40-fold or 240-fold increased mutant frequencies above NER⁻ or NER⁺ background levels, respectively, in crosses with NER⁻ females. With decreasing dose, / ratios decreased from 9 to 14 at high doses down to ≈1 at the two lowest doses, indicating that a small fraction of premutagenic lesions induced by EO cannot be repaired by the NER system of Drosophila. Linear extrapolation from high to low EO exposure led to an underestimation of the mutation frequency actually observed at low doses. The pattern of EO-induced ring chromosome loss (CL) differed in two respects from that observed for forward mutations: (a) an increase in CL frequencies was observed only at the two highest EO exposure levels, and (b) inactivation of the NER pathway by the mus201 mutant had no measurable effect on the occurrence of CL. The absence of a potentiating effect of mus201 on EO-induced clastogenicity suggests the formation of clastogenic DNA lesions not causing point mutations, and which are not repaired by NER. Consistent with an inversed correlation of reactivities towards N7-guanine and chain length of 1,2-alkyl-epoxides, the relative mutagenic efficiencies of EO:PO:BO are 100:7.2:1.8 for the NER⁺ groups, and 100:20:0.7 in the absence of NER. Although in Drosophila germ cells EO is also more effective as a clastogen than PO, the difference (EO:PO=100:58) is much smaller than for recessive mutations. These results provide another argument that DNA lesions generating base substitutions as opposed to those causing clastogenic damage may not be the same for these agents.     

In vitro reaction of ethylene oxide with DNA and characterization of DNA adducts.

Ethylene oxide (EO) is a direct-acting SN2 alkylating agent and a rodent and probable human carcinogen. In vitro reactions of EO with calf thymus DNA in aqueous solution at neutral pH and 37 degrees C for 10 h resulted in the following 2-hydroxyethyl (HE) adducts (nmol/mg DNA): 7-HE-Gua (330), 3-HE-Ade (39), 1-HE-Ade (28), N6-HE-dAdo (6.2), 3-HE-Cyt (3.1), 3-HE-Ura (0.8) and 3-HE-dThd (2.0). Reference (marker) compounds were synthesized from reactions of EO with 2'-deoxyribonucleosides and DNA bases, isolated by paper and high performance liquid chromatography and characterized on the basis of chemical properties and UV, NMR and mass spectra. In agreement with our earlier studies with propylene oxide (PO) (Chem.-Biol. Interact., 67 (1988) 275-294) and glycidol (Cancer Biochem. Biophys., 11 (1990) 59-67), alkylation at N-3 of dCyd by EO under physiological conditions resulted in the rapid hydrolytic deamination of 3-HE-dCyd to 3-HE-dUrd. The hydroxyl group on the alkyl side chain which forms after epoxide alkylation is mechanistically involved in this rapid hydrolytic deamination. These results may provide important insights into the mechanisms of mutagenicity and carcinogenicity exhibited by EO and other SN2 aliphatic epoxides.     

Unscheduled DNA synthesis correlated to alkylation of hemoglobin in individuals occupationally exposed to propylene oxide

Exposure to propylene oxide was determined previously by the degree of alkylation of hemoglobin measured on the histidine residue as N-3-(2-hydroxypropyl) histidine, using blood samples from 8 propylene oxide-exposed employees and 13 unexposed referents. Mononuclear leukocytes isolated from the same blood samples were used to quantify DNA repair proficiency following an in vitro challenge with the carcinogen, N-acetoxy-2-acetylamino-fluorene. Decreases in the DNA repair proficiency index correlated significantly to in vivo exposure levels to propylene oxide (r = –0.64, p <0.03). These data suggest a possible short-term biological assay for monitoring the in vivo genotoxic effects of propylene oxide exposure in the human population.Abbreviations EO ethylene oxide - NA-AAF N-acetoxy-2-acetylaminofluorene - HOPrHIS N-3-(2-hydroxypropyl) histidine - PO propylene oxide - UDS unscheduled DNA synthesis     

Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

The research background for risk assessment of ethylene oxide: aspects of dose

Data for relationships between in vivo doses inferred from levels of hemoglobin (Hb) or DNA adducts and administered (by inhalation or injection) doses of ethylene oxide (EO) in mice, rats and humans are reviewed. At low absorbed doses or dose rates these relationships appear to be linear, whereas at higher dose rates deviations from linearity due to saturation kinetics of detoxification and of DNA repair as well as certain toxic effects have to be allowed for. If these factors are taken into consideration, a rather consistent picture is obtained for animal studies, with a variation by less than a factor 2 between estimates of adduct level increments or in vivo dose increments per unit of administered dose. Although the value for in vivo dose per unit of exposure dose (ppm-hour) in humans is uncertain because of unreliable data for the time-weighted average exposure level, the most likely value for this relationship, supported by data for ethene, agrees with data for the rodents. In the animal species testis doses are approximately one-half of the blood doses inferred from Hb adducts.     

Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver

Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.     

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.     

Thermodynamic analysis of micellization in PEO–PPO–PEO block copolymer solutions from the hydrogen bonding point of view

A thermodynamic approach is suggested to study the micellization mechanism of poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO) triblock copolymers solutions from the hydrogen bonding point of view. Using Flory–Huggins theory, an association model is presented to describe hydrogen bonded (HB) chains, which are bridged by hydrogen bonds between water molecules and segments of the copolymer. The entropic change due to hydrogen bonding is formulated and the individual contribution of EO–water (EO–W) and PO–water (PO–W) hydrogen bonding to the micellization are derived respectively. Fourier transform infrared (FTIR) spectroscopy is applied to obtain the information of hydrogen bonds. During the temperature-dependent micellization of P105 block copolymer solutions, rapid disruption of PO–W hydrogen bonds is observed by FTIR and the calculated entropy relating to PO–W hydrogen bonds increases drastically compared with that of EO–W hydrogen bonds. The results demonstrate that PO–W hydrogen bonds play a dominant role in micellization.     

Alkylation of DNA and hemoglobin in the mouse following exposure to propene and propylene oxide

Male CBA mice were exposed to propene, unlabelled or 14C-labelled, by inhalation, or to 14C-labelled propylene oxide by intraperitoneal injection. 2-Hydroxypropyl adducts to guanine-N-7 in DNA of various organs and to N-terminal valine and histidine-N pi in hemoglobin were measured. The adduct levels observed show that propylene oxide is the major primary metabolic product of propene. A direct comparison of propylene oxide with the homologous compound ethylene oxide on the basis of adduct levels introduced (in DNA and in hemoglobin) at equimolar injected amounts, shows that propylene oxide is 6-10 times less effective than ethylene oxide.     

Polymer recycling in aqueous two-phase extractions using thermoseparating ethylene oxide–propylene oxide copolymers

This is a study on the recovery and recycling of copolymer in aqueous two-phase systems containing random copolymers of ethylene oxide (EO) and propylene oxide (PO). The random copolymers separate from water solution when heated above the lower critical solution temperature (LCST). The primary phase systems were composed of EOPO copolymer and hydroxypropyl or hydroxyethyl starch. After phase separation the upper EOPO phase was removed and subjected to temperature induced phase separation. Copolymers with different EO/PO compositions have been investigated, EO50PO50 [50% EO and 50% PO (w/w)], EO30PO70 and EO20PO80. The temperature required for thermoseparation decreases when the PO content of the copolymer is increased. The effect on the recovery of copolymer after addition of salts, a second polymer or protein was investigated. The added components increased the recovery of copolymer after thermoseparation, e.g., increased the amount copolymer separated from the water phase after thermoseparation. Recycling of copolymer and measurements of polymer concentrations in the primary top and bottom phases after repeated recycling steps was performed. The fluctuation in polymer concentration of the phases was very small after recycling up to four times. Partitioning of the proteins BSA and lysozyme was studied in primary phase systems after recycling of copolymer. The partition coefficients of total protein and lysozyme was not significantly changed during recycling of copolymer. More than 90% of the copolymer could be recovered in the thermoseparation step by optimising the temperature and time for thermoseparation. In repeated phase partitionings in EOPO–starch systems the EO50PO50 copolymer could be recovered to 77% including losses in primary system and thermoseparation, which is equivalent to a total copolymer reuse of 4.3 times.