Pediatric reference intervals for endocrine markers in healthy children and adolescents on the Liaison XL (DiaSorin) immunoassay system.

OBJECTIVES: Prominent physiological changes occurring throughout childhood and adolescence necessitate the consideration of age and sex in biomarker interpretation. Critical gaps exist in pediatric reference intervals (RIs) for specialized endocrine markers, despite expected influence of growth and development. The current study aimed to establish and/or verify RIs for six specialized endocrine markers on a specialized immunoassay system. METHODS: Samples were collected from healthy children and adolescents (5 to <19 years) and apparently healthy outpatients (0 to <5 years) as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER). Serum samples were analysed for aldosterone, renin (plasma), thyroglobulin, anti-thyroglobulin, growth hormone, and insulin-like growth factor-1 (IGF-1) on the Liaison XL (DiaSorin) immunoassay platform. RIs (2.5th and 97.5th percentiles) were established for aldosterone, renin, thyroglobulin, anti-thyroglobulin, and growth hormone. Manufacturer-recommended pediatric RIs for IGF-1 were verified. RESULTS: Age-specific RIs were established for aldosterone, renin, and thyroglobulin, while no age-specific differences were observed for anti-thyroglobulin or growth hormone. IGF-1 was the only endocrine marker studied that demonstrated significant sex-specific differences. Manufacturer-recommended IGF-1 RIs were verified for children aged 6 to <19 years, while those for children aged 0 to <6 years did not verify. CONCLUSIONS: This study marks the first time that pediatric RIs for aldosterone and renin were established in the CALIPER cohort and highlights the dynamic changes that occur in water and sodium homeostasis during the first years of life. Overall, these data will assist pediatric clinical laboratories in test result interpretation and improve clinical decision-making for patients tested using Liaison immunoassays.

Early determinants of atherosclerosis in paediatric systemic lupus erythematosus.

OBJECTIVES: To assess traditional and non-traditional cardiovascular risk factors and to determine the prevalence and correlates of early vascular markers of atherosclerosis in paediatric systemic lupus erythematosus (pSLE). METHODS: Fifty-four adolescents with pSLE had cardiovascular risk factor assessment, disease activity and vascular testing including carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD), arterial stiffness measures, and myocardial perfusion studies. RESULTS: The traditional risk factors of hypertension, elevated triglycerides, apolipoprotein B, haemoglobin A1c and insulin levels and non-traditional risk factors of elevated homocysteine and fibrinogen were present (all p<0.001). Some arterial stiffness measures, central pulse wave velocity and characteristic impedance were elevated (p<0.001), but CIMT, FMD and myocardial perfusion were normal. Cumulative prednisone dose correlated with total cholesterol (r=0.5790, p<0.001) and elevated LDL-C (r=0.4488, p=0.0012). Hydroxychloroquine treatment correlated negatively with total cholesterol (r=-0.4867, p=0.0002), LDL-C (r=-0.4805, p=0.0002) and apolipoprotein B (r=-0.4443, p=0.0011). In multivariate analysis LDL-C correlated with cumulative prednisone dose and negatively with hydroxychloroquine treatment (R2=0.40, p<0.001). CONCLUSIONS: An increased burden of traditional and non-traditional risk factors and early evidence of insulin resistance and increased central arterial stiffness were present in paediatric SLE. Disease-specific and therapy-related factors are likely modifying these cardiovascular risk profiles warranting prospective longitudinal studies.

Continuous bupivacaine infusion post-iliac crest bone graft harvesting in pediatric cleft surgery: role and comparison with ketorolac.

OBJECTIVE: To investigate the use of intravenous ketorolac and iliac crest bupivacaine infusion in the management of iliac crest donor-site pain in the pediatric cleft population. The null hypothesis was there is no difference with respect to pain scores between ketorolac and iliac crest bupivacaine infusion as analgesic adjuncts to intravenous opioids. METHODS: A total of 54 children and adolescents (27 boys, 27 girls) undergoing alveolar cleft repair or Le Fort I osteotomy were assigned randomly in a prospective, single-blinded fashion to one of three groups: intravenous ketorolac plus iliac crest normal saline infusion, intravenous ketorolac plus iliac crest bupivacaine infusion, or iliac crest bupivacaine infusion alone. Iliac crest infusions and ketorolac were administered for 48 hours or until discharge, whichever occurred first. All patients received morphine via a patient-controlled analgesia device. MAIN OUTCOME MEASURE(S): Primary outcome was pain score, and secondary outcomes were morphine consumption and satisfaction scores. RESULTS: Pain scores, morphine consumption, and satisfaction scores were not significantly different among groups. Estimated costs were significantly higher for bupivacaine infusion than intravenous ketorolac. CONCLUSIONS: Iliac crest donor-site pain is well managed in this patient population. Intravenous ketorolac and iliac crest bupivacaine infusion provide comparable analgesia for iliac crest bone graft donor-site pain in children and adolescents.

The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. METHODS: Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. RESULTS: Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. CONCLUSIONS/INTERPRETATION: Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: evidence for PKC activation and oxidative stress-activated signaling pathways.

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine(307) phosphorylation of IRS-1. IRS-1 Serine(307) phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IkappaB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKalpha/beta, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.

Pediatric reference intervals for calculated LDL cholesterol, non-HDL cholesterol, and remnant cholesterol in the healthy CALIPER cohort.

OBJECTIVES: Increased prevalence of pediatric obesity and associated co-morbidities has heightened the concern for cardiovascular disease (CVD) risk later in life. Although the fasting lipid profile is traditionally used to assess CVD risk, the non-fasting lipid profile may simplify lipid testing and better predict CVD risk. Unfortunately, non-fasting lipid reference values are limited, particularly for children. The Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) has recruited thousands of healthy pediatric subjects to develop a pediatric reference interval database. Here, CALIPER reports pediatric reference intervals for non-fasting calculated low-density lipoprotein cholesterol (LDLc), non-high-density lipoprotein cholesterol (non-HDLc) and remnant cholesterol. METHODS: Non-fasting serum samples from the CALIPER cohort of community children and adolescents were previously analyzed for HDLc, total cholesterol, and triglycerides. These values were used to calculate LDLc, non-HDLc, and remnant cholesterol and subsequently establish reference intervals with corresponding 90% confidence intervals according to CLSI EP28-A3c guidelines. Reference intervals were also calculated using alternative statistical methods highlighted in recent literature. RESULTS: All three lipid parameters required an age partition at 1 year due to wider reference intervals in the first year of life. LDLc and non-HDLc required sex partitioning for subjects 1-<10 years. Non-HDLc upper reference limit was higher than the 2011 National Heart, Lung, and Blood Institute (NHLBII) pediatric recommended cut-offs, suggesting elevated atherogenic lipoproteins in a proportion of apparently healthy pediatric subjects. The LDLc upper reference limit (10-<19 year partition) was the same as the NHLBI cut-off, potentially due to lower calculated LDLc values in the non-fasting state. CONCLUSIONS: With the increased use of non-fasting lipid profiles, age- and sex-specific reference intervals and appropriate clinical decision limits are necessary for pediatric lipid monitoring. Our data supports the notion that appropriate decision limits, rather than reference intervals, should be used to interpret lipid levels in children as there is a high prevalence of hyperlipidemia in the apparently healthy pediatric population.

Intracellular degradation of apolipoprotein B generates an N-terminal 70 kDa fragment in the endoplasmic reticulum.

Regulated apoB degradation in HepG2 cells occurs in the endoplasmic reticulum (ER), is catalyzed by an N-acetylleucylleucylnorleucinal (ALLN)-sensitive proteinase, and generates a specific 70 kDa fragment (Adeli, K., 1994, J. Biol. Chem. 269, 9166-9175) [corrected]. In the present report, we have characterized the 70 kDa fragment by immunoprecipitation of permeabilized HepG2 cells with a battery of monoclonal antibodies against various sites on the apoB molecule. N-Terminal monoclonal antibodies (1D1 and 2D8) were capable of binding to the 70 kDa fragment suggesting that this polypeptide is an N-terminal fragment of the intact apoB. Subcellular fractionation of permeabilized cells and carbonate extraction resulted in the detection of the 70 kDa fragment in the ER lumen. Endoglycosidase H treatment confirmed that the fragment is N-linked glycosylated. We hypothesize that the ALLN-sensitive proteinase which may be located on the luminal side of the ER membrane, catalyzes an initial cleavage of apoB near the N-terminus generating a 70 kDa fragment, which is then released into the ER lumen.

Mechanisms of hepatic very low-density lipoprotein overproduction in insulin resistance.

An important complication of insulin-resistant states, such as obesity and type 2 diabetes, is an atherogenic dyslipidemia profile characterized by hypertriglyceridemia, low plasma high-density lipoproteins (HDL) cholesterol and a small, dense low-density lipoprotein (LDL) particle profile. The physiological basis of this metabolic dyslipidemia appears to be hepatic overproduction of apoB-containing very low-density lipoprotein (VLDL) particles. This has focused attention on the mechanisms that regulate VLDL secretion in insulin-resistant states. Recent studies in animal models of insulin resistance, particularly the fructose-fed hamster, have enhanced our understanding of these mechanisms, and certain key factors have recently been identified that play important roles in hepatic insulin resistance and dysregulation of the VLDL secretory process. This review focuses on these recent developments as well as on the hypothesis that an interaction between enhanced flux of free fatty acids from peripheral tissues to liver, chronic up-regulation of de novo lipogenesis by hyperinsulinemia and attenuated insulin signaling in the liver may be critical to the VLDL overproduction state observed in insulin resistance. It should be noted that the focus of this review is on molecular mechanisms of the hypertriglyceridemic state associated with insulin resistance and not that observed in association with insulin deficiency (e.g., in streptozotocin-treated animals), which appears to have a different etiology and is related to a catabolic defect rather than secretory overproduction of triglyceride-rich lipoproteins.

Secretion of apolipoprotein B in serum-free cultures of human hepatoma cell line, HepG2.

We have developed a defined medium which can maintain efficient growth of HepG2 cells sustaining the synthesis of a variety of plasma proteins including apolipoprotein B. This defined system was used to investigate long-term effects of insulin, estrogen, triiodothyronine, cholesterol, and oleate on the growth pattern of HepG2 cells and secretion rate of apolipoprotein B. Oleate and triiodothyronine caused significant increases in secretion of apolipoprotein B. The stimulatory effect of triiodothyronine was only observed after long (6 days) exposure of cells to the hormone. In contrast, insulin caused up to a 4-fold decrease in the secretion rate of apolipoprotein B during the early growth periods. This inhibitory effect appeared to be partially abolished after 6 days. Our data suggest that some important questions on regulation of apolipoprotein B expression can be addressed by the long-term culture of HepG2 cells in defined medium.

Clinical laboratory investigation of the Sanofi ACCESS CK-MB procedure and comparison to electrophoresis and Abbott IMx.

This evaluation was undertaken to verify the application protocol for the CK-MB assay on the ACCESS Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Chaska, MN). The results show that the ACCESS CK-MB assay total imprecision was 6.8% to 9.1%. Analytical linearity of the ACCESS CK-MB assay was excellent in the range of < 1-214 micrograms/L. A comparison of the ACCESS CK-MB assay with the IMx (Abbott Laboratories, Abbott Park, IL) method shows good correlation r = 0.990 (n = 108). Linear regression analysis yielded Y = 1.36X-0.3, Sx/y = 7.2. ACCESS CK-MB values also correlated well with CK-MB by electrophoresis with r = 0.968 (n = 132). The linear regression equation for this comparison was Y = 1.08X + 1.4, Sx/y = 14.1. The expected non-myocardial infarction range of CK-MB determined by the ACCESS system was 1.3-9.4 micrograms/L (mean = 4.0, n = 58). The ACCESS CK-MB assay would appear to be rapid, precise and clinically useful.

Regulation of apolipoprotein B biogenesis in human hepatocytes: posttranscriptional control mechanisms that determine the hepatic production of apolipoprotein B-containing lipoproteins.

OBJECTIVES: Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins appears to be a common cause of hyperlipoproteinemia in humans. Patients with overproduction states secrete denser cholesterol ester-rich lipoprotein particles which are highly atherogenic. The formation of apoB particles involves a very complex process that requires the coordinated synthesis and assembly of apoB, triglycerides, cholesterol esters, phospholipids, and other components. ApoB expression is an important prerequisite for the assembly and secretion of apoB particles. Evidence to date appears to suggest that apoB expression is regulated posttranscriptionally. ApoB secretion rate is determined at the levels of apoB translocation into the endoplasmic reticulum (ER) as well as degradation within the ER. RESULTS AND HYPOTHESIS: Based on available data, we postulate that the rate of apoB particle secretion is determined at the critical point where newly-synthesized apoB interacts with core lipids, particularly triglycerides. The supply of these lipids determines the rate of translocation of the apoB molecule across the ER membrane and into the ER lumen. Lipidation of apoB facilitates its proper folding, its assembly into a lipoprotein particle, and its extracellular secretion. In the absence of lipids, apoB is misfolded resulting in the abortion of ER translocation and subsequent degradation by an apoB specific protease. CONCLUSIONS: The balance between intracellular degradation and extracellular secretion determines the rate at which the human liver secretes apoB particles.

Human bioassays to assess environmental genotoxicity: development of a DNA repair assay in HepG2 cells.

A direct assessment of the effects of environmental chemicals on human health has been hampered by the lack of suitable experimental systems. We have recently employed a human liver cell line (HepG2) to assess the biological effects of pollutants at both cellular and DNA levels. A Neutral Red dye uptake assay was used to assess potential cytotoxic effects of xenobiotics. DNA damage was quantified using an unscheduled DNA synthesis assay that measures repair that is induced following exposure to genotoxic compounds. HepG2 cells responded to the known mutagens, 4-nitroquinoline N-oxide and methylmethane sulfonate, both in the Neutral Red assay for cytotoxicity and two DNA repair assays for genotoxicity (monitored autoradiographically or by liquid scintillation counting). The HepG2 DNA repair and cytotoxicity assays also responded to an extract (containing polycyclic aromatic hydrocarbons) of sediment obtained from a polluted site in the Great Lakes. Results indicate that this system can be deployed further to assess potential cyto- and genotoxicity of pollutants. The development of human cell culture assays is a critical step towards a full assessment of the risk that such pollutants pose to human health.

Apolipoprotein B gene polymorphism and plasma lipids and lipoproteins in a Canadian Caucasian population.

We have investigated the frequency of Hind III DNA polymorphism of the human apolipoprotein B gene in a Canadian Caucasian population with coronary artery disease, as documented by angiography, and a healthy control population. Patients had significantly (p < 0.05) higher levels of cholesterol, triglycerides, LDL-cholesterol, apolipoprotein B, and lower level of apoAI compared to the controls. Restriction fragment-length polymorphism analysis detected nine hybridizable fragments denoted as H1 to H9. The H1, H2, H3, and H7 alleles were polymorphic. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1-H9] genotype was most frequently observed in the patients (frequency = 0.68). We were unable to strongly associate any of the alleles or genotypes detected with the changes in lipids. The additional alleles observed in the patient group may indicate possible mutations at the 3' end of the apolipoprotein B gene locus.

Tocotrienol: a review of its therapeutic potential.

OBJECTIVES: To summarize new knowledge surrounding the physiological activity of tocotrienol, a natural analogue of tocopherol. RESULTS: The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. In the vitamin E group, alpha-tocopherol is considered to be the most active form. However, recent research has suggested tocotrienol to be a better antioxidant. Moreover, tocotrienol has been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein B and lipoprotein(a) plasma levels. In addition, tocotrienol has been suggested to have an anti-thrombotic and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. CONCLUSION: The physiological activities of tocotrienol suggest it to be superior than alpha-tocopherol in many situations. Hence, the role of tocotrienol in the prevention of cardiovascular disease and cancer may have significant clinical implications. Additional studies on its mechanism of action, as well as, long-term intervention studies, are needed to clarify its function. From the pharmacological point-of-view, the current formulation of vitamin E supplements, which is comprised mainly of alpha-tocopherol, may be questionable.

Human bioassays to assess environmental genotoxicity: development of a DNA break bioassay in HepG2 cells.

OBJECTIVES: Increasing interest in environmental health issues has created a demand for improved methods for the assessment of pollutant effects on humans. Our laboratory has developed an in vitro assay for the quantification of genotoxicity, monitored as DNA single strand breaks (SSB), in the HepG2 human hepatoma cell line. DESIGN AND METHODS: This assay procedure, which is based upon alkaline unwinding and hydroxylapatite DNA chromatography, is both rapid and simple to perform. RESULTS: HepG2 cells responded to the standard mutagen, 4-nitroquinoline N-oxide, demonstrating SSB formation at concentrations above 0.1 mumol/L. Phenanthrene-9,10-quinone, a component of diesel exhaust, mediated SSB formation at concentrations above 250 nmol/L. Finally, an extract of contaminated sediment from the Great Lakes Basin mediated SSB formation in a dose-dependent manner. CONCLUSIONS: These results illustrate the utility of this human genotoxicity assay for future use in screening of environmental pollutants.

A competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100.

OBJECTIVES: We have developed a competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100 (apoB) and conducted a preliminary evaluation of the method. DESIGN AND METHODS: In the assay, apoB-rabbit immunoglobulin G (IgG) conjugate competes with apoB in the sample for binding to acridinium N-hydroxysuccinimide labelled anti-apoB antibody. Goat anti-rabbit-IgG immobilized to magnetic particles is used to separate the apoB-rabbit IgG conjugate bound to the labelled anti-apoB antibody. Chemiluminescence, measured using a Ciba Corning Magic-Lite chemiluminometer, is inversely proportional to the concentration of apoB in the sample. RESULTS: The assay is completed in 1-2 h. Within-run imprecision (n = 5) determined at 10, 100, and 200 micrograms/L of apoB was found to be 4.8%, 2.4%, and 3.5%, respectively. The between-run imprecision (n = 5) at the same concentrations of apoB was determined to be 14.2%, 9.5%, and 7.7%, respectively. The proposed assay showed reasonable correlation (r = 0.86) with a commercially available immunoturbidimetric method. The lower limit of detection was 2.2 micrograms/L (4.0 pmol/L). CONCLUSIONS: This assay may be useful in tissue culture studies where sensitive measurement of secreted and intracellular concentrations of apoB are required. Our preliminary evaluation of the assay appears to confirm is usefulness.

Influence of DT diaphorase on quinone-mediated genotoxicity in human and fish cell lines.

The influence of the quinone-reducing enzyme, DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase], on the genotoxicity of quinones was examined in two cell lines, namely a human hepatoma cell line, HepG2 and a brown bullhead fibroblast cell line, BB. The quinone-reductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic reductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model quinones, menadione (MND) and 9,10-phenanthrenequinone (PQ) was examined in an alkaline unwinding assay for DNA single-strand breaks. Results revealed that DT diaphorase was the predominant quinone reductase in cytosols of both cell lines, and that levels of specific DT diaphorase activity were generally equivalent in the two species. Despite these similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor, dicoumarol, HepG2 cells exhibited a marked exacerbation of genotoxicity in the presence of either MND or PQ, indicating protective influence of the enzyme. In contrast, quinone genotoxicity in BB cells was not affected by DT diaphorase inhibition, indicating the lack of a protective effect of DT diaphorase. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities, and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium.

We have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.

In vitro toxicological methods for environmental health testing.

Increasing public interest in environmental health issues has created a demand for alternatives to using animals for assessing the toxic effects of chemical mixtures on humans. This review focuses on applications of in vitro toxicological screening methods developed for human health biomonitoring using cultured clonal cell lines, which have the which have the following advantages: genetic variation between samples and experiments is minimal; the cultivation of cell lines is rapid and consistent conditions for culture are easily maintained; most of the phenotypic variation that is encountered with use of cell donors is eliminated; and radiolabeled precursors can be used for labeling and quantifying protein and DNA. We describe the current state of development of in vitro toxicity testing methods, present detailed procedures for the test methods optimized in our laboratory, and compare these techniques with other approaches. Toxicity testing using cell lines provides a mechanism to quantify the risks associated with environmental exposure to chemical mixtures.