7-Ethoxycoumarin and 7-ethoxyresorufin O-deethylase in human foetal and adult liver: studies with monoclonal antibodies

The 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylase activities were investigated in the microsomal fractions from 5 human adult and 3 foetal livers and 5 human foetal adrenals. The enzyme activity expressed as pmol/min. per mg microsomal protein was higher with 7-ethoxyresorufin as substrate in all investigated specimens with average values (+/- S.E.M.) of 74 +/- 27, 13 +/- 3 and 12 +/- 1 in adult and foetal livers and foetal adrenals, respectively. Monoclonal antibodies raised against 3-methylchloranthrene or phenobarbital induced rat liver cytochrome P-450 were investigated with respect to their inhibiting effects on the rate of O-deethylation of both substrates in human adult liver. Only the monoclonal antibody against the 3-methylcholanthrene induced cytochrome P-450 inhibited the O-deethylation of 7-ethoxyresorufin to 64 to 79 percent of control values. The other antibody had no effect on this or the other O-deethylase activity. Thus, the 7-ethoxyresorufin O-deethylase is partly catalyzed in human adult liver by a cytochrome with an epitope that is recognized by the monoclonal antibody against 3-methylcholanthrene induced rat liver cytochrome P-450. With foetal liver the low activity of the enzyme became unmeasurable in the presence of this antibody.     

The effect of cigarette smoking on 7-ethoxyresorufin O-deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies.

Four cytochrome P-450 enzyme activities, 7-ethoxyresorufin O-deethylase (ERDE), coumarin 7-hydroxylase (CH), 7-ethoxycoumarin O-deethylase (ECDE) and aryl hydrocarbon hydroxylase (AHH) were measured in human liver needle biopsy samples from smokers and non-smokers. Cigarette smoking was verified and quantitated by measuring plasma cotinine levels. Enzyme inhibitory monoclonal antibodies (MAb) to a 3-methylcholanthrene-induced (MAb 1-7-1) and phenobarbitone-induced (MAb 2-66-3) rat hepatic cytochrome P-450 were used to measure the contribution of MAb-defined, epitope-specific cytochromes P-450 to the total reaction measured for each of the above activities. ERDE activity was significantly elevated in the livers of cigarette smokers, whereas AHH, CH or ECDE activities were not affected by cigarette smoking. No correlation was observed between plasma cotinine concentration and ERDE activity. MAb 1-7-1 inhibited hepatic ERDE activity to a variable extent (from 0 to 65%), but had very little or no effect on AHH, CH or ECDE activities. The inhibitory effect of MAb 1-7-1 on ERDE activity was greater than 50% in the non-smokers. MAb 2-66-3 had no inhibitory effect on any of the enzyme activities studied. In contrast to liver both ERDE and AHH on human placental microsomes from cigarette smokers were inhibited by MAb 1-7-1. The MAb 2-66-3 was without effect. Cigarette smoking induces a form of P-450 in human liver, responsible for ERDE activity, that contains an epitope recognized by MAb 1-7-1. This form of cytochrome P-450 is insensitive to MAb 2-66-3 and is not contributing to AHH, CH or ECDE activities of human liver.     

Selective induction and inhibition of the components of 7-ethoxycoumarin O-deethylase activity in the rat

1. Treatment of rats with 3-methylcholanthrene increased V_max of the high-affinity component of 7-ethoxycoumarin O-deethylase activity 60-fold. There was also an increase in V_max of the low-affinity component.

2. Treatment with phenobarbitone increased V_max of the high-affinity component sixfold whilst not affecting the K_m of this component. Modest changes were also observed in the kinetics of the low-affinity component.

3. Following treatment with 3-methylcholanthrene, the sensitivity of both the high- and low-affinity components of activity to inhibition by α-naphthoflavone was considerably increased, with the IC₅₀ decreasing from >250μM to < 10 μM in both instances.

4. Following treatment with phenobarbitone, the sensitivity of the low-affinity component to inhibition by metyrapone was considerably increased, with the IC₅₀ decreasing from > 1000 μM to 96 μM. There was also a modest, but significant, increase in sensitivity of the high-affinity component to metyrapone.

5. These results indicate that both components of 7-ethoxycoumarin O-deethylase activity are catalysed by more than one form of cytochrome P-450.     

Selective induction and inhibition of the components of 7-ethoxycoumarin O-deethylase activity in the rat

Treatment of rats with 3-methylcholanthrene increased Vmax of the high-affinity component of 7-ethoxycoumarin O-deethylase activity 60-fold. There was also an increase in Vmax of the low-affinity component. Treatment with phenobarbitone increased Vmax of the high-affinity component six-fold whilst not affecting the Km of this component. Modest changes were also observed in the kinetics of the low-affinity component. Following treatment with 3-methylcholanthrene, the sensitivity of both the high- and low-affinity components of activity to inhibition by alpha-naphthoflavone was considerably increased, with the IC50 decreasing from greater than 250 microM to less than 10 microM in both instances. Following treatment with phenobarbitone, the sensitivity of the low-affinity component to inhibition by metyrapone was considerably increased, with the IC50 decreasing from greater than 1000 microM to 96 microM. There was also a modest, but significant, increase in sensitivity of the high-affinity component to metyrapone. These results indicate that both components of 7-ethoxycoumarin O-deethylase activity are catalysed by more than one form of cytochrome P-450.     

Critical evaluation of 7-ethoxycoumarin O-deethylase activity measurement in intact isolated rat hepatocytes

In the determination of 7-ethoxycoumarin O-deethylase activity in intact isolated rat hepatocytes various factors influence the assay, including: the decay of 7-ethoxycoumarin fluorescence which is temperature and pH dependent; the measured fluorescence which has to be adjusted for the inner filter effect; glucose addition to the medium which influences the activity; all organic solvents which inhibit the enzymic activity, with dimethylformamide provoking the smallest effect (partial competitive inhibition); the enzymic reaction which is inhibited by the product of reaction; and the presence of bovine serum albumin in the medium which affects the enzymic activity. Biphasic kinetics are observed for the O-deethylation of ethoxycoumarin in intact isolated rat hepatocytes. For the high-affinity component, Km and Vmax values are 5 microM and 43 pmol/min X 10(6) cells and for the low-affinity component are 414 microM and 915 pmol/min X 10(6) cells. Addition of the substrate in dimethylformamide or omitting bovine serum albumin from the medium cause important changes in these kinetic parameters.     

The effect of glucose, insulin and dexamethasone upon 7-ethoxycoumarin O-deethylase activity of adult rat hepatocytes in primary culture

The influence of glucose, insulin and/or dexamethasone on the 7-ethoxycoumarin O-deethylase activity of rat hepatocytes in primary culture was investigated. The addition of extra glucose to the medium attenuated the fall in enzyme activity observed during the first 24 h in culture, this effect being dose-dependent. The inclusion of insulin further enhanced this effect, but glucose + insulin did not prevent the decrease in enzyme activity in the subsequent 48 h. The inclusion of dexamethasone, alone or in combination with glucose + insulin, also attentuated the initial decline in enzyme activity. After 24 h in culture, the enzyme activity increased such that after 72 h in culture, the activity was greater than that measured in the freshly isolated cells.     

The effect of glucose,insulin and dexamethasone upon 7-ethoxycoumarin O-deethylase activity of adult rat hepatocytes in primary culture

1. The influence of glucose, insulin and/or dexamethasone on the 7-ethocycoumarin O-deethylase activity of rat hepatocytes in primary culture was investigated.

2. The addition of extra glucose to the medium attenuated the fall in enzyme activity observed during the first 24 h in culture, this effect being dose-dependent. The inclusion of insulin further enhanced this effect, but glucose — insulin did not prevent the decrease in enzyme activity in the subsequent 48 h.

3. The inclusion of dexamethasone, alone or in combination with glucose + insulin, also attentuated the initial decline in enzyme activity. After 24 h in culture, the enzyme activity increased such that after 72 h in culture, the activity was greater than that measured in the freshly isolated cells.     

Two different 7-ethoxycoumarin O-deethylase activities in mice after pretreatment with sulmazole and cobaltous chloride

Pretreatment of male C57BL/6J Han mice with 200 mg of sulmazole (AR-L 115 BS) daily for 2 days results in an up to 4fold increase of the 7-ethoxycoumarin O-deethylase. The resulting effect was compared to that after a pretreatment with cobaltous chloride which also leads to a 4fold increase. Enzyme kinetic parameters of the 7-ethoxycoumarin O-deethylase are different for the two inducers in respect to the affinity for the substrate, but the inhibitions by metyrapone and alpha-naphthoflavone are similar. Comparison of the microsomal protein patterns after partial purification of microsomes revealed totally different patterns after sulmazole and cobaltous chloride. Sulmazole produces a 54 kDa protein band and cobaltous chloride a band at 48.5 kDa. Therefore it is concluded that in mice at least two 7-ethoxycoumarin O-deethylase activities can be induced. The electrophoretic pattern after sulmazole is different from those after 3-methylcholanthrene and isosafrole. This was also proven by enzyme kinetic investigations with ethoxyresorufin as substrate.     

Histological, growth and 7-ethoxyresorufin O-deethylase (EROD) activity responses of greenback flounder Rhombosolea tapirina to contaminated marine sediment and diet

Pathological abnormalities and mixed function oxygenase (MFO) enzyme changes are frequently used as indicators of anthropogenic contaminant exposure and effect. However, there is a paucity of research investigating the effects of contaminated sediment on native Australian benthic teleosts. As part of an ecotoxicological assessment of contaminated marine sediments in northern Tasmania, CYP1A induction, histological and growth response of the greenback flounder, Rhombosolea tapirina, exposed to contaminated marine sediments were examined. Hatchery reared flounder were exposed to reference sediment, contaminated sediment or contaminated sediment and diet for 6 weeks. CYP1A induction, using the ethoxyresorufin-O-deethylase (EROD) assay, and the histological and growth response in the flounder were examined on cessation of the exposure trial. Significant differences were found between treatments in histological, growth and EROD response. Exposure to contaminated sediment and diet elicited a multi-organ histological response: principally partial and total epidermal erosion and multifocal necrosis of the liver. The prevalence of total epidermal erosion was greatest with exposure to disturbed contaminated sediment (66.65+/-16.65%). The prevalence of multifocal necrosis of the liver was greatest with exposure to contaminated sediment and diet (66.65+/-16.65%). Growth reduction, measured as percentage growth inhibition, was evident in flounder exposed to contaminated sediment and diet (18.2+/-11.99%). Additionally, exposure to contaminated sediment and diet elicited elevated induction of the EROD liver detoxification enzyme (139.65+/-24.22 pmol/min/mg protein) compared to exposure to contaminated sediment and non-contaminated diet (6.25+/-0.81 pmol/min/mg) indicating the presence and potential bioavailability of xenobiotics via food. Further, more inhibited growth and histological alteration associated with exposure to contaminated sediment and diet suggest contaminants in Deceitful Cove sediment are cytotoxic.     

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin by rat and hairless mouse skin strips

The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was studied in rat and hairless mouse skin strips. These preparations supported de-ethylation, sulphation and glucuronidation reactions. The de-ethylation reaction was inducible in both species by pretreatment with either 5,6-benzoflavone or 3-methylcholanthrene. The hairless mouse strips exhibited a greater basal de-ethylase activity than rat strips, although the latter was the more responsive to inducers. Accompanying the increase in de-ethylation activity was a change in the pattern of metabolites, with a large increase in the percentage of the unconjugated metabolite. When 7-hydroxycoumarin was employed as the primary substrate the glucuronide was the major metabolite formed by strips from both species. The glucuronidation and sulphation activities were unchanged by 3-methylcholanthrene pretreatment.     

The metabolism of 7-ethoxycoumarin in primary maintenance cultures of adult rat hepatocytes

Summary 7-Ethoxycoumarin is metabolized to 7-hydroxycoumarin in short-term (1–4 days) maintenance cultures of adult rat hepatocytes. The 7-hydroxycoumarin is predominantly found as the sulphate and glucuronic acid conjugates. This pattern of metabolism is very similar to that observed with freshly-isolated rat hepatocytes and suggests that the culture system may be of value in studying the metabolism of novel chemicals designed for human therapeutic use. The specific activity of microsomal monooxygenase activity falls by 50–60% during 4 days in culture. This is not reflected by the sulphate and glucuronic acid conjugation pathways which are retained at normal levels throughout the entire 4-day culture period.     

Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions

Human and rat liver microsomal fractions exhibit non-linear Michaelis-Menten kinetics in the O-deethylation of both phenacetin and 7-ethoxycoumarin. Comparison of various models indicated that the data were best described by a biphasic plot, which could be interpreted in terms of two populations of cytochrome P-450. The K(m)'s of the high affinity phase of 7-ethoxycoumarin O-deethylase activity were 1.8 +/- 0.4 microM and 2.3 +/- 0.4 microM for human and rat respectively while the K(m)'s of the low affinity phase were 205 +/- 20 microM and 237 +/- 59 microM in the two species respectively. V(max) of the high affinity phase of human 7-ethoxycoumarin O-deethylase activity was 96.9 +/- 19.0 pmol mg(-1) min(-1) and the activity of the corresponding phase in the rat was 2.7 times greater. The activities of the low affinity phase were 10-15 times greater than the respective activity of the high affinity phase. Rat and human also had similar values for the K(m)'s of the two phases of phenacetin O-deethylase activity, around 5 microM for the high affinity phase and 300 microM for the low affinity phase. Total activity was very similar in the two species, 1500-1750 pmol mg(-1) min(-1) and the difference between the two phases of activity was only 2.5-fold in man and 10-fold in rat. Studies on the effects of the in vitro modifiers of monooxygenase activity alpha-naphthoflavone and metyrapone further supported the hypothesis that the two phases of O-deethylase activity represent two different forms or populations of cytochrome P-450.     

The effect of Aroclor 1254 pretreatment on the phase I and phase II metabolism of 7-ethoxycoumarin in isolated viable rat kidney cells

The metabolism of 7-ethoxy-and 7-hydroxy-coumarin was studied in viable kidney cortex cells isolated from control and Aroclor 1254-pretreated rats. Such pretreatment led to induction of the microsomal mono-oxygenase-mediated Phase I system but did not produce induction of Phase II glucuronidation or sulphation activity. Induction of the microsomal mono-oxygenase system led to an increase in the amount of unconjugated Phase I metabolite present during sequential Phase I and Phase II metabolism. It is suggested that this increase is due to some form of ‘uncoupling’ of the microsomal mono-oxygenase and glucuronidation systems.     

单克隆抗体治疗自身免疫性疾病的研究进展

自身免疫性疾病的治疗通常采用的糖皮质激素、免疫抑制剂等,虽然有一定疗效,但长期使用都会产生严重的不良反应,而且都只能减缓病情的发展,并不能根治疾病。近10余年来,单克隆抗体治疗自身免疫性疾病已成为研究热点。这些单克隆抗体通过不同的机制发挥作用,包括结合可溶性细胞因子和生长因子,调节受体和受体信号,以及通过抗体介导的细胞依赖的细胞毒作用、抗体介导的细胞吞噬作用、补体依赖的细胞毒作用耗竭异常的免疫细胞及介导细胞信号等。研制更高效、更安全、给药更方便的抗体药物是未来抗体药物的发展趋势。通过改造IgG的氨基酸序列或者对其蛋白质进行一些修饰,以及以多个不同的信号途径或者致病介质为靶标的双特异性抗体,是新型单克隆抗体药物研究的重要方向。本文就治疗自身免疫性疾病的单克隆抗体研究进展作一综述。     

Current and future issues in the manufacturing and development of monoclonal antibodies

Despite a slow beginning, monoclonal antibodies have had many successes over the past decade. It is important that these successes continue, bringing more products for more indications to market. Although manufacturing is not the most common cause of product failure, product quality issues can delay antibody development. Manufacturing has depended on the triad of process validation, process control and product testing. Applying product knowledge proactively to manufacturing (quality by design) may allow greater flexibility and maintain or improve product quality. An integrated approach to biological characterization is an important aspect of product knowledge. Greater product knowledge also facilitates development in other disciplines. Independent of manufacturing strategy, there are a number of regulatory hurdles in initial and ongoing antibody development. These are described to help prevent unnecessary delays.     

Activities in chick embryos of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon (benzo[a]pyrene) hydroxylase and their induction by 3,3',4,4'-tetrachlorobiphenyl in early embryos

Basal activities of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon (benzo[a]pyrene) hydroxylase were determined in whole-liver homogenates from chick embryos of different ages, from newly hatched chicks and from chicks a few days old. The enzyme activities increased substantially in chick embryo livers between days five and 10, remained at a fairly constant level until day 19, and reached a peak in activity one day after hatching. The optimal pH value was lower than 7.0 for both enzyme activities. The total increase in activity from day five to one day after hatching was about 300-fold for 7-ethoxycoumarin O-deethylase and about 75-fold for aryl hydrocarbon (benzo[a]pyrene) hydroxylase. Treatment of eggs with 3,3',4,4'-tetrachlorobiphenyl resulted in increased metabolism of both substrates by five-day-old chick embryo livers. The increase in aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity was 14-fold while that of 7-ethoxycoumarin O-deethylase was approximately double.