Drug Metab Dispos-1997-Raucy-1429-35

HUMAN LYMPHOCYTE CYTOCHROME P4502E1,A PUTATIVE MARKER FOR ALCOHOL-MEDIATED CHANGES IN HEPATIC CHLORZOXAZONE ACTIVITY

JUDY L.RAUCY,ERIC D.SCHULTZ,MIKE R.WESTER,SANJEEV ARORA,DAVID E.JOHNSTON,JOHN L.OMDAHL,

AND

SUSAN P.CARPENTER

The Agouron Institute (J.L.R.,E.D.S.,M.R.W.,S.P.C.)and Department of Internal Medicine,Division of Gastroenterology (S.A.,D.E.J.),and

Department of Biochemistry (J.L.O.),University of New Mexico School of Medicine

(Received April 29,1997;accepted August 5,1997)

ABSTRACT:

Cytochrome P450(CYP)2E1is implicated in a variety of chemically initiated hepatotoxicities,including alcoholic liver disease.These pathological conditions arise from increased production of reac-tive intermediates caused by elevated enzyme concentrations.Thus,the ability to detect enhanced CYP2E1levels would aid in identifying individuals at high risk for xenobiotic-promoted liver injury.With this in mind,the present investigation assessed in vivo chlorzoxazone metabolism and compared pharmacokinetic pa-rameters with CYP2E1expression in blood.Twenty-two subjects were recruited and divided into two groups,control subjects and alcohol abusers,based on responses to two screening question-naires.Those individuals with higher survey scores,i.e.those who consumed alcohol more frequently,exhibited higher rates of chlor-zoxazone metabolism.Indeed,a correlation (r ?0.66,p <0.01)was obtained when scores were compared with the pharmacokinetic

parameter AUC for chlorzoxazone.Lymphocyte microsomes iso-lated from blood samples obtained from these same individuals were subjected to immunoblot analyses to detect CYP2E1levels.That lymphocytes contained CYP2E1was confirmed by reverse transcription-polymerase chain reaction and sequence analysis of the cDNA.Quantification of immunoreactive bands revealed that levels of this P450were 2.3-fold higher in alcoholics than in control subjects.This increase in lymphocyte CYP2E1content in alcoholic subjects coincided with a 2.1-fold increase in chlorzoxazone clear-ance and a 2-fold decrease in the AUC for chlorzoxazone.Impor-tantly,a correlation (r ?0.62,p <0.01)was observed between CYP2E1content in lymphocytes and chlorzoxazone clearance rates.Thus,monitoring lymphocyte CYP2E1expression may pro-vide a substitute for estimating hepatic activity of this P450.

The ethanol-inducible P4501CYP2E1is a toxicologically impor-tant enzyme because of its unique ability to convert many substrates to cytotoxins (1,2).Moreover,chemical metabolism by this enzyme produces oxygen radicals,which can ultimately lead to lipid peroxi-dation (3–6).Ethanol is one such substrate,and its oxidation by CYP2E1has been implicated in alcoholic liver disease (7,8).Of particular concern is that CYP2E1concentrations in humans vary extensively,due in part to pathophysiological conditions (including obesity)and exposure to xenobiotics (such as ethanol or isoniazid)(9–11).Variations in the expression of the enzyme caused by these factors determine the degree of hepatotoxicity elicited by alcohol or other chemicals.

CYP2E1is primarily an hepatic enzyme;however,evidence sug-gests that it is also present in other tissues,including lung,kidney,nasal mucosa,and bone marrow (12–14).More recently,CYP2E1was

found in the white cell fraction of peripheral blood from humans (15),rabbits (16),and rats (17).Expression of CYP2E1in the lymphocyte fraction of white blood cells appears to be influenced by the same factors that affect the concentration of the hepatic enzyme,including xenobiotics and physiological states (15–17).Indeed,insulin-depen-dent diabetes enhances the expression of lymphocyte CYP2E1in humans (15),and starvation increases the expression of CYP2E1protein and mRNA in rodent lymphocytes (17).With regard to xeno-biotics,ethanol produced a 10-fold enrichment of lymphocyte CYP2E1in rabbits,compared with their control counterparts (16).Interestingly,CYP2E1induction by either ethanol (16)or fasting (17)occurred in both liver and lymphocytes,in a parallel manner.Thus,changes in CYP2E1content in white blood cells may reflect xenobi-otic-promoted alterations in other tissues,including liver.

Because expression of CYP2E1is altered by many factors affecting chemical metabolism and therefore toxicity,the ability to easily estimate human CYP2E1content is appealing,particularly for assess-ment of risks from chemical exposure.Currently,the concentrations of hepatic CYP2E1can be estimated by determining pharmacokinetic parameters of CZX metabolism.However,application of this proce-dure may not be suitable for screening large numbers of samples in general clinical settings.Therefore,approaches that are less time consuming and more convenient are currently being investigated.One approach could include estimating hepatic CYP2E1content from its expression in HPBLs.This may be feasible,given results obtained in animal studies (16,17).Although several investigations have demon-strated xenobiotic-metabolizing P450enzymes,including CYP1A1(18),3A5(19),and 2E1(15),in human white blood cells,there are no

This work was supported by United States Department of Health and Human Services Grants AA08990(J.L.R.)and NCRR-GCRC RR00997(Clinical Research Center,University of New Mexico).Approval for studies involving human subjects was granted by the University of New Mexico Human Research Review Commit-tee (HRRC 90–213).1

Abbreviations used are:P450or CYP,cytochrome P450;CZX,chlorzoxa-zone;6-OH-CZX,6-hydroxychlorzoxazone;AU,absorbance units;bp,base pairs;CAGE,cut,annoyed,guilty,and eye opener;NLAES,National Longitudinal Alco-hol Epidemiological Survey;TBST,Tris-buffered saline with Tween-20;HPBL,human peripheral blood lymphocyte;BAC,blood alcohol concentration.Send reprint requests to:Dr.Judy L.Raucy,The Agouron Institute,505Coast Blvd.South,Suite 400,La Jolla,CA 92037-4696.

0090-9556/97/2512-1429–1435$02.00/0D RUG M ETABOLISM AND D ISPOSITION

Vol.25,No.12Copyright ?1997by The American Society for Pharmacology and Experimental Therapeutics

Printed in U.S.A.

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direct comparisons with the hepatic content of these P450s.The present investigation was designed to determine whether CYP2E1levels in human lymphocytes are higher in alcoholic subjects and whether white cell expression reflects hepatic CZX activity.

Materials and Methods

Volunteers.Twenty-two subjects (10men and 12women,ranging in age from 20to 50years)participated in this investigation.Volunteers were healthy subjects within 20%of ideal body weight.Individuals with diabetes,individ-uals receiving prescription medications,or individuals diagnosed with a code-pendency were eliminated from the study.At the time of recruitment,informed consent was obtained,questionnaires were completed,and a blood sample was drawn for routine blood evaluations (Reference Laboratory,Inc.,Albuquerque,NM).Based on laboratory results,subjects demonstrating abnormal liver function values or those diagnosed with hepatic disease were discharged.Volunteers proceeding with the investigation were admitted to the University of New Mexico Clinical Research Center,where they received thorough physical examinations.Subjects remained overnight in the Clinical Research Center to prevent alcohol abusers access to alcoholic beverages and to allow BACs in individuals with elevated levels to return to zero.BACs of ?10mg/dl ensured that competitive inhibition of CZX metabolism did not occur.During the overnight stay,volunteers were placed on caffeine-free diets and alcoholics were carefully monitored for any signs of withdrawal.At 7:30a.m.,blood samples were obtained to determine BACs;all subjects possessed values of ?5mg/dl.Blood (320ml)was drawn at 8:00a.m.for lymphocyte isolation from all but two alcoholic subjects;in these two cases the large sample was unobtainable.After the blood drawing,500mg of CZX was orally adminis-tered.Blood samples (10ml)were subsequently obtained at 0,0.25,0.50,0.75,1.0,1.5,2,3,4,5,6,8,10,and 12hr.Plasma was separated by centrifugation at 2300rpm for 15min at 4°C,immediately frozen,and stored at ?20°C until assayed.

Clinical Evaluations.Subject screening procedures consisted of two types of evaluations.First,subjects completed a survey developed by our laboratory,composed of questions adapted from the CAGE questionnaire,a common screening instrument used by health care professionals to detect alcohol problems (20,21).Second,a more detailed personal interview was used to compile alcohol usage profiles for all individuals.The profile incorporated physiological,psychological,and social components and was determined by posing questions compiled in the NLAES.The NLAES is an Alcohol Use Disorders and Associated Disabilities Interview Schedule questionnaire similar to that in Diagnostic and Statistical Manual of Mental Disorders ,fourth edition,but it was developed primarily for epidemiological research purposes (22).Both surveys were subjectively scored using a protocol developed by an epidemiologist in our laboratory.

Responses to the questionnaire adapted from the CAGE questionnaire were subjectively assigned weighted scores,with greater weight being given to questions that were more specific.The emphasis of the survey was on the frequency of drinking and the amount of alcohol consumed.A simple yes/no question was given less weight than a question specifically asking how frequently the behavior was exhibited.Responses to how often the subject drank alcoholic beverages were scored from 0to 2,with 0being never,not in the last year,or less than 1day/month.A score of 1was indicative of behavior that occurred either 1–3days/month or 1day/week,and a score of 2indicated that alcohol was consumed 2–3days/week,4–5days/week,or every day.Greater weight was placed on a score of 2when summing the raw scores for each question.

The NLAES had three variables for each question that dictated the raw score.The first variable determined whether the subject had ever experienced the event or circumstance stated in the question.For an affirmative response a score of 1was assigned,whereas a negative response was given a score of 0.The second variable assessed whether the experience had occurred in the last 12months.The third established a weighted factor for how frequently the circumstance had occurred during the last 12months and ranged from 1to 5,with 1being almost never and 5being all the time.If the event had not happened in the last 12months,then the weighted score was assigned a value of 0.The score for a given question was equal to the sum of the first and third variables.The second variable (whether the experience had occurred in the last

12months)was used as an introduction to a more specific investigation of behavior frequency and as an indicator of whether a weighted factor was present.The total score of the survey was the sum of the raw scores for each question.The severity of behavior was quantified by summing the total scores for the questionnaire developed by our laboratory and the NLAES.

Lymphocyte Isolation.Lymphocytes were immediately separated from 320ml of whole blood according to the method of Boyum (23).Briefly,20ml of Histopaque-1077(Sigma Chemical Co.,St.Louis,MO)was added to a 50-ml conical centrifuge tube.Whole blood was layered onto Histopaque-1077,and tubes were centrifuged at 400g for 30min at ambient temperature.After centrifugation,the opaque interface was transferred to a new tube,phosphate-buffered saline was added,and tubes were centrifuged at 250g for 10min at 4°C.Cells were resuspended in RPMI 1640medium (Gibco BRL Products,Gaithersburg,MD)containing 100units/ml penicillin,100?g/ml streptomycin,and 10%fetal calf serum and were cultured overnight to separate lymphocytes from monocytes.Cultures were then transferred to 50-ml conical tubes and centrifuged at 250g for 10min at 4°C.The lymphocyte pellet was either resuspended in 5.0ml of homogenizing buffer (0.1M Tris-HCl,pH 7.4),containing 0.5mM phenylmethylsulfonyl fluoride (Sigma),for microsome preparation or mixed with Trizol (Gibco BRL Products)for RNA isolation.Both samples were frozen in liquid nitrogen and stored at ?70°C until use.Microsome Preparation.Microsomes were isolated from white cells by previously published procedures (16,24).Briefly,lymphocytes were sonicated on ice for 3?60sec to disrupt cells.Microsomes were then prepared from disrupted cells,and pellets were resuspended in sucrose assay buffer (25).Lymphocyte microsomes were stored at ?70°C until use.Protein values were determined with bovine serum albumin as a standard (26).

Immunoblot Analysis.Microsomal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose filters.Filters were subsequently blocked in 5%nonfat dry milk/TBST (20mM Tris buffer,pH 7.6,137mM NaCl,0.1%Tween-20)for 1hr at 37°C and allowed to react overnight at 4°C in 5%milk/TBST containing a previously characterized anti-human CYP2E1IgG (5?g/ml)(27).Filters were then incubated for 60min with biotinylated goat anti-rabbit IgG (1:2000in TBST;Calbiochem,La Jolla,CA),followed by a 60-min incubation with streptavidin-conjugated horseradish peroxidase (1:2000in TBST;Calbiochem)at room temperature.Immunochemical staining was performed by reaction of the filters with 10ml of enhanced chemilumi-nescence detection reagents (Amersham,Arlington Heights,IL)for 1min at room temperature and exposure to Amersham Hyperfilm for 10–30sec.Immunoreactive CYP2E1content in lymphocyte microsomes was quantified with a Microtek Scanmaker IIHR scanner interfaced to ImageQuant software.Lymphocyte microsomal protein was applied in various amounts (5–75?g)to determine the linear range of signal intensity of immunoblots.The concentra-tion of microsomal protein (25?g)used for all subsequent immunoblot analyses was within the linear portion of that curve.

Reverse Transcription/Polymerase Chain Reaction.Total RNA from lymphocytes was isolated using Trizol reagent (Gibco BRL Products)and quantified by measurement of its absorbance at 260nm;purity was assessed by determination of the 260/280-nm ratio.First-strand cDNA synthesis was performed with RNA as previously described (25).The cDNA was then amplified using oligonucleotide primers that were 21bp in length and flanked CYP2E1exons 4(bp 501–523)and 6(bp 954–976),as described elsewhere (25).The resulting DNA was ligated into pCRII vector (Invitrogen,San Diego,CA)and transformed into competent (INV ?F ?)Escherichia coli cells.Ultra-pure plasmid DNA was isolated from the E.coli transformants and sequenced using the dideoxy-chain termination method (28)and Sequenase (United States Biochemicals,Cleveland,OH).

Analytical Methods.CZX and 6-OH-CZX concentrations in plasma were determined by a reverse-phase HPLC-based assay (9).Plasma samples (250?l)were thawed on ice,and 750?l of 2M sodium acetate buffer,pH 4.5,was added.Each sample was evaluated in triplicate.Twenty microliters (200activity units)of Helix pomatia type H-2?-glucuronidase (Sigma)were added to each tube and incubated overnight at 37°C.After incubation,200?l of 0.2M theophylline was added to each tube,for use as an internal standard.The reaction was terminated by addition of 3ml of 1N HCl and centrifuged at 3500g for 10min at 4°C.The supernatant was removed and placed in separate tubes containing 2ml of ethyl acetate.Samples were vigorously vortex-mixed

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for 1min and centrifuged at 2000rpm for 8min at 4°C.The organic layer was removed and the aqueous phase was subjected to a second extraction with 2ml of ethyl acetate.The extracts were pooled and evaporated to dryness under nitrogen with low heat.The residues were resuspended in 100?l of mobile phase (20%,v/v,acetonitrile/80%,v/v,0.5%phosphoric acid)and vigorously vortex-mixed.A 20-?l aliquot of extract was subsequently analyzed by use of a model 504autosampler (Beckman Instruments,Palo Alto,CA),a model 600solvent delivery system (Waters Millipore,Milford,MA),and a model 486detector (Waters Millipore)set at 287nm.Chromatographic separation was achieved with a C 8Ultrasphere column (5-?m,4.6?250mm;Beckman Instruments).The mobile phase was pumped at a flow rate of 1ml/min.Standard curves of area ratios for CZX/theophylline and 6-OH-CZX/theophylline were constructed from plasma containing various amounts (0.20–8.0?g/ml)of CZX (Sigma)and 6-OH-CZX (McNeal Consumer Products,Fort Washington,PA).Recoveries of 6-OH-CZX and CZX were 77and 70%,respectively,with this extraction method.Values for each sample were extrap-olated from linear calibration curves (r ?0.99)for the standards.The interday coefficient of variation (N ?22),determined by addition of CZX and 6-OH-CZX to plasma samples,was approximately 15%for each at concentrations of 2,4,and 8?g/ml.The limits of detection for both CZX and 6-OH-CZX were 0.09?0.01?g/ml.

Pharmacokinetic Analysis.The AUC for CZX and its metabolite were determined using the trapezoid rule.Noncompartmental analysis was used to compare the oral clearance of CZX in control and alcoholic subjects and was determined from the ratio of the administered dose to the total AUC.

Statistical Analysis.Three separate statistical tests were used to determine significance or possible correlations between study populations.Group com-parisons were performed using Student’s t test for independent samples.A one-way analysis of variance was used to determine any statistical significance that might exist between more than two distributions or sample groups.Correlation analyses were also performed,and a curve fit determined the association of the two fields.Statistical calculations were made using Graph-Pad Instat version 2.05a software.Statistical significance was set at p ?0.05.Values were expressed as the mean ?SE.

Results

Volunteers were randomly recruited with strategically placed fly-ers.The mean age of the 10male subjects involved was 33.1?3.16years,and that of the 12female subjects was 36.67?2.49years.Participants were divided into two groups,control subjects and alco-hol abusers,based on subjective scores received in the screening surveys.For the 22individuals participating in this investigation,scores ranged from 0to 192(fig.1).The distribution demonstrates that control subjects possessed scores from 0to 38,whereas alcohol abusers exhibited scores from 58to 192.Significant differences in scores based on gender,between alcoholic men (114.4?24.20)and women (99.17?14.56)or between control male (7.6?2.34)and female (4.00?1.24)volunteers,were not apparent,suggesting that alcohol consumption did not exhibit a gender preference.Body weights (men,75.36?4.73kg;women,62.58?3.02kg)and heights (men,179.25?3.53cm;women,160.20?1.46cm)for all subjects were within the normal range for healthy individuals.

Subjects were given a 500-mg single dose of CZX to assess CYP2E1-mediated metabolism in vivo .Pharmacokinetic parameters for CZX metabolism were determined for each of the volunteers.The mean plasma concentration-time profiles for all control subjects and alcohol abusers are shown in fig.2.In control subjects,CZX was rapidly absorbed,with plasma concentrations peaking at 2hr and declining to detection limits by 12hr.Plasma concentrations of the metabolite also peaked at 2hr in control subjects but declined to undetectable levels after 10hr.In contrast,plasma concentrations of CZX in alcohol abusers (fig.2B )peaked at 1.5hr but at 1.4-fold lower concentrations than observed in plasma from control subjects.The concentration of 6-OH-CZX in alcohol abusers also exhibited a peak at 1.5hr,but the content was 1.4-fold greater than that observed in

samples from control subjects.Furthermore,by 10hr the parent drug had declined to nearly undetectable levels in alcoholic subjects.These results indicated that peak plasma concentrations of both the parent drug and its metabolite were achieved earlier in alcohol abusers than in control subjects and that CZX levels declined at a faster rate in alcoholics.Additionally,greater plasma concentrations of 6-OH-CZX were obtained in the alcohol abusers,compared with control subjects.Analysis of pharmacokinetic parameters indicated that the C max of CZX was 1.4-fold lower in alcohol abusers than control subjects,whereas the C max for 6-OH-CZX was 1.4-fold higher in alcoholics (table 1).As a result of lower plasma CZX concentrations in alcohol-ics,oral clearance was significantly increased by 100%(table 1).No statistically significant differences were noted between alcoholics and control subjects in the AUC for 6-OH-CZX.However,there was a significant difference in the AUC for CZX between control subjects (24.64?2.49)and alcohol abusers (12.56?1.11).Furthermore,the 6-OH-CZX/CZX AUC ratio was 2.2-fold greater in samples from alcohol abusers than in those from control subjects (table 1).Param-eters were also compared by gender,but no significant differences were observed for CZX clearance or AUC for CZX (table 1).Results indicate that chronic alcohol consumption influences pharmacokinetic parameters for CZX.Indeed,when the AUC for CZX was compared with the frequency of alcohol consumption,as determined by the survey score,a correlation was observed (r ?0.66,p ?0.01)(fig.3).The relationship between the CYP2E1-mediated activity of liver,as determined by in vivo CZX metabolism,and the concentration of the enzyme in HPBLs was explored.Detection of CYP2E1in lympho-cytes was performed by immunoblot analysis of isolated microsomes.Before examination of individual samples,varying amounts (5–125?g)of lymphocyte microsomal protein were applied to polyacryl-amide gels and,after separation,transferred to a nitrocellulose filter,which was stained with anti-human CYP2E1IgG.Fig.4is a repre-sentative immunoblot containing microsomes from lymphocytes and liver,for comparison.A band migrating 3500Da below than that of liver is apparent in lanes containing lymphocyte microsomes.The faster migration of the lymphocyte protein may be due to differences in molecular mass between the liver and blood enzymes.A standard curve was constructed by application of 5–75?g of

lymphocyte

F I

G .1.Distribution of survey scores.

The 22subjects participating in this investigation were assigned survey scores based on responses to two questionnaires,as described in Materials and Methods .The scores indicate the frequency and amount of alcohol consump-tion.

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microsomal protein and subsequent quantification of the 50,500-Da immunoreactive band.From this curve,25?g was selected for use on subsequent blots,because this amount provided a readily detectable signal within the linear range of intensity (fig.5).

To verify that CYP2E1was present in HPBLs,we performed reverse transcription/polymerase chain reaction.Total RNA isolated from lymphocytes of four subjects was used as a template in a reverse transcription reaction to generate cDNA.The cDNA was then mixed with specific primers,as described in Materials and Methods,and amplified for 30cycles by the polymerase chain reaction.Amplifica-tion yielded a 475-bp fragment from lymphocyte RNA that was similar in size to the fragment amplified from a human liver RNA sample.Sequence analysis revealed that the amplified lymphocyte DNA was identical to the corresponding region of human CYP2E1cDNA previously described by Song et al.(29).

Quantification of immunoblots containing lymphocyte microsomes from 20subjects in this study revealed a 10.5-fold variability,which ranged from 7.5to 80AU/?g microsomal protein.A 2.3-fold greater staining intensity was observed in lymphocyte microsomes from alcoholic subjects (42.57?9.05AU/?g),compared with control subjects (18.14?3.04AU/?g)(fig.6).Additionally,a 2.1-fold

increase in CZX clearance and a 2-fold decrease in the AUC for CZX were noted in alcoholic subjects,compared with control subjects (table 1and fig.6).To assess whether expression of CYP2E1in lymphocytes could serve as an indicator of P450activity in liver,enzyme levels were compared with CZX metabolism.Correlation analysis was performed on immunoquantified lymphocyte CYP2E1and the pharmacokinetic parameter of CZX clearance.Interestingly,a correlation (r ?0.62,p ?0.01)was observed between these two parameters (fig.7),suggesting that chronic alcohol consumption enhanced expression of the enzyme in liver and white cells in a parallel fashion.

Discussion

The present study compared the expression of the ethanol-inducible P450CYP2E1in HPBLs with in vivo activity of the hepatic enzyme.The 6-hydroxylation of CZX was used as a biomarker for hepatic CYP2E1activity,because several investigations (30–33)have vali-dated its specificity for this reaction in vivo .Other P450enzymes,including recombinant human CYP1A2and rat CYP3A,appear to exhibit CZX 6-hydroxylase activity (34,35).However,the in vivo contribution of recombinant CYP1A2and the clinical significance of rat CYP3A to CZX hydroxylation have not been confirmed.Indeed,reports suggest that neither CYP1A2nor CYP3A4plays a major role in this reaction in human subjects.One such study showed that disulfiram,a specific CYP2E1inhibitor,significantly decreased me-tabolism of CZX in vivo when simultaneously administered to vol-unteers (33).Furthermore,increased CZX metabolism in alcoholics,compared with control subjects (36,37),suggests that altered phar-macokinetic parameters are due to ethanol-mediated induction of a P450,presumably CYP2E1,because this enzyme is the major ethanol-inducible P450in humans (2,38).That CYP2E1is the primary P450induced by chronic alcohol exposure was recently challenged,when it was determined that CYP3A4levels in isolated hepatocytes were increased by ethanol (39).However,confirmation of this induction in vivo remains to be established.At present,CZX appears to be a valid estimate of human CYP2E1activity in vivo,and chronic alcohol consumption increases its metabolism via enhanced expression of this enzyme.

CYP2E1content in HPBLs from subjects involved in the present study was assessed by immunoblot analyses.The blots revealed a band in HPBL microsomes that migrated below that in liver (fig.4).Presently,the 3500-Da difference in the molecular masses of lym-phocyte and hepatic CYP2E1is believed due to either variations in the amount of microsomal protein used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis or structural alterations between the enzymes from the two tissues.Suggestive of the latter argument,previous studies from our laboratory demonstrated that rabbit micro-somal CYP2E1in lymphocytes and liver are the same molecular weight (16),despite the fact that 40-fold more protein from lympho-cyte microsomes was applied to the gel.The amounts of liver and lymphocyte microsomal protein loaded onto the gels in the current investigation were similar to those used in the previous study.Further evidence suggesting a structural difference in CYP2E1of blood and liver was provided by a study where HPBL homogenates from dia-betic children exhibited a lower molecular weight form of CYP2E1than the corresponding enzyme in human hepatic microsomes (15).Taken together,evidence supports the contention that structural dif-ferences may exist between the human hepatic and blood enzymes.Despite the lower molecular weight of CYP2E1in lymphocytes,enhanced expression of the enzyme was observed in alcoholic sub-jects.Indeed,the extent of CYP2E1induction was similar to the increase in CZX metabolism,demonstrating good agreement

with

F I

G .2.Plasma concentration-time profiles for CZX and 6-OH-CZX.Plasma concentration-time curves were constructed for control subjects (N ?11)(A )and alcohol abusers (N ?11)(B )after a single 500-mg oral dose of CZX.Each value represents the mean ?SE.F ,CZX;f ,6-OH-CZX.

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studies conducted in experimental animals (16,17).However,in the present study the magnitude of induction of white cell CYP2E1in alcoholics was less than observed in rabbits treated with ethanol (16).This lower level of CYP2E1enhancement may be due to the addi-tional overnight culturing of HPBLs,coupled with the rapid turnover of this enzyme.Recombinant human CYP2E1transfected into Hep G2cells displayed a half-life in culture of 2.5–6hr,whereas in the presence of inducing agents,including ethanol,the recombinant en-zyme exhibited a half-life of 37hr (40).Additionally,results obtained

in vivo demonstrated that the 6-OH-CZX/CZX ratio decreased rapidly during ethanol withdrawal in alcoholic subjects (37).This rapid de-cline in the ratio was attributed to a 2.5-day half-life determined for CYP2E1.Thus,if HPBL CYP2E1possesses characteristics similar to those of its recombinant or hepatic counterpart,quantification of the ethanol-enhanced enzyme in HPBLs occurred here before turnover,but when concentrations were declining.

Results (data not shown)from our laboratory indicated that CYP2E1content was reduced by approximately 20%in microsomes from white cells cultured overnight,compared with noncultured cells of alcoholic subjects.The advantage gained by culturing white cells overnight is that monocytes are separated from lymphocytes (41).Elimination of monocytes,where CYP2E1is absent,provides a more homogeneous cell population,thereby increasing immunoblot sensi-tivity.Thus,the advantage of increased sensitivity by elimination of monocytes surpassed the decrease in alcohol-induced CYP2E1con-tent caused by the 12-hr overnight culture.

TABLE 1

Pharmacokinetic parameters for CZX metabolism were assessed for each individual by quantifying CZX and its metabolite,6-OH-CZX,after oral administration of 500mg.Quantification was by an HPLC-based assay described in Materials and Methods.Values are mean ?SE of three separate

determinations.

Subject

Clearance

CZX C max

6-OH-CZX C max

CZX AUC

6-OH-CZX AUC

6-OH-CZX/CZX AUC Ratio

liters/hr ?g/ml ?g/ml ?g/ml/hr ?g/ml/hr Male control (5)a

21.31?2.74 5.59?0.580.47?0.0925.48?4.04 2.83?0.240.12?0.01Female control (6)23.31?3.65 6.12?0.380.46?0.0323.94?3.39 3.22?0.160.15?0.02Control (11)

22.40?2.26 5.89?0.320.43?0.0224.64?2.49 3.04?0.150.13?0.01Male alcohol abuser (5)47.60?14.58 4.60?0.560.62?0.1013.11?2.14 3.19?0.290.31?0.10Female alcohol abuser (6)43.20?4.41 4.18?0.780.56?0.0912.11?1.18 2.95?0.200.26?0.04Alcohol abuser (11)

45.29?6.65b 4.37?0.48b 0.58?0.07b 12.56?1.11b 3.06?0.10

0.29?0.05b

a Values in parentheses are the number of subjects in each group.b

Statistically significant difference from control,p ?

0.05.

F I

G .3.Correlation between AUC for CZX values and survey scores.Correlation analysis was performed on the AUC for CZX values and the survey scores for 22subjects involved in this study.A correlation coefficient of r ?0.66(p ?0.01)was

obtained.

F I

G .4.Representative immunoblot of various concentrations of lymphocyte

microsomal protein.Microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter,and the filter was reacted with anti-human CYP2E1IgG,as described in Materials and Methods .Lanes 1–9,5,10,15,20,25,50,75,100,and 125?g of lymphocyte microsomal protein;lane 10,human liver microsomes (0.5?g of

protein).

F I

G .5.CYP2E1content in various protein concentrations of lymphocyte

microsomes.

Lymphocyte microsomes were prepared from one individual and subjected to immunoblot analysis as described in Materials and Methods .Various amounts of microsomal protein (5,10,20,25,50,and 75?g)were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,followed by trans-fer to a nitrocellulose filter and staining with anti-human CYP2E1IgG.The human anti-CYP2E1-reactive band was quantified as described.Values for CYP2E1content are expressed as AU and are the means of two determina-tions.

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In summary,this investigation demonstrated that CYP2E1protein and its corresponding mRNA were present in freshly isolated HPBL microsomes.Before this report,CYP2E1was identified in whole homogenates of HPBLs cultured for 4days (15).Eliminating the 4-day culture period simplified the procedure and reduced the time required for detecting CYP2E1in human blood samples.Importantly,the increase in CYP2E1protein in lymphocytes was similar to the alterations in CZX clearance and the AUC for CZX,indicating that chronic ethanol consumption produced similar increases in CYP2E1expression in lymphocytes and liver.Thus,P450enzymes expressed in HPBLs may be used to predict alterations in hepatic P450-mediated

activity caused by xenobiotic exposure.Screening of P450concen-trations in freshly isolated HPBLs,rather than with invasive liver biopsies or time-consuming in vivo metabolic studies,may be an efficient method for identifying individuals at greater risk for chem-ically promoted hepatotoxicity.

Acknowledgments.The authors thank Gerald Curley,Barbara Mounho,and Margo Lopez for their technical assistance.

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药学英语第五版第三单元

Biochemistry Seeks to Explain Life in Chemical Terms The molecules of which living organisms are composed conform to all the familiar laws of chemistry, but they also interact with each other in accordance with another set of principles, which we shall refer to collectively as the molecular logic of life. These principles do not involve new or yet undiscovered physical laws or forces. Instead, they are a set of relationships characterizing the nature, function, and interactions of biomolecules. If living organisms are composed of molecules that are intrinsically inanimate, how do these molecules confer the remarkable combination of characteristics we call life? How is it that a living organism appears to be more than the sum of its inanimate parts? Philosophers once answered that living organisms are endowed with a mysterious and divine life force, but this doctrine (vitalism) has been firmly rejected by modern science. The basic goal of the science of biochemistry is to determine how the collections of inanimate molecules that constitute living organisms interact with each other to maintain and perpetuate life. Although biochemistry yields important insights and practical applications in medicine, agriculture, nutrition, and industry, it is ultimately concerned with the wonder of life itself. All Macromolecules Are Constructed from a Few Simple Compounds Most of the molecular constituents of living systems are composed of carbon atoms covalently joined with other carbon atoms and with hydrogen, oxygen, or nitrogen. The special bonding properties of carbon permit the formation of a great variety of molecules. Organic compounds of molecular weight less than about 500, such as amino acids, nucleotidase, and monosaccharide, serve as monomeric subunits of proteins, nucleic acids, and polysaccharides,

药学专业英语药学词汇

6-磷酸葡萄糖脱氢酶 glucose-6-phosphate dehydrogenase Janbon综合症 Janbon's syndrome PPB浓度 parts per billion concentration pphm浓度 parts per hundred million concentration PPH浓度 parts per hundred concentration ppm浓度 parts per million concentration 安全范围 safety range 安全试验法 innocuity test method 安全系统 safety coefficient 安慰剂 placebo 螯合剂 chelating agent 靶细胞 target cell 白蛋白微球制剂 albumin microballoons 百分浓度 percentage concentration 半合成抗生素 semisynthetic antibiotics 半抗原 haptene 半数致死剂量 half lethal dose ; median lethal dose; LD50 半衰期 half-life period; half life time 包衣片 coated tablet 薄膜衣 film-coating 饱和溶液 saturated solution 贝克勒尔 Becquerel 被动免疫 passive immunity 被动转运 passive transport

崩解度 disintegration 崩解剂 disintegrants 必需氨基酸 essential aminoacid 必需脂肪酸 essential fatty acid 变态反应 allergy; allergic reaction 表面活性 surface activity 表面张力 surface tension 丙种射线 gamma rays 补体 complement 补体系统 complement system 不良反应 adverse reaction 不完全抗原 incomplete antigen 搽剂 liniments 长期毒性实验 long term toxicity test 长效制剂 prolonged action preparation 肠肝循环 enterohepatic circulation 肠溶控释片 enteric controlled release tablets 肠溶衣 enteric coating 处方 prescription;recipe 穿透促进剂 penetration enhancers 磁性控释制剂 magnetic controlled release dosage form 磁性药物制剂 magnetic medicinal preparations 大分子 macromolecule 单克隆抗体 monoclonal antibody

药学英语第五版原文翻译 (2)(2020年7月整理).pdf

Introduction to Physiology Introduction Physiology is the study of the functions of living matter. It is concerned with how an organism performs its varied activities: how it feeds, how it moves, how it adapts to changing circumstances, how it spawns new generations. The subject is vast and embraces the whole of life. The success of physiology in explaining how organisms perform their daily tasks is based on the notion that they are intricate and exquisite machines whose operation is governed by the laws of physics and chemistry. Although some processes are similar across the whole spectrum of biology—the replication of the genetic code for or example—many are specific to particular groups of organisms. For this reason it is necessary to divide the subject into various parts such as bacterial physiology, plant physiology, and animal physiology. To study how an animal works it is first necessary to know how it is built. A full appreciation of the physiology of an organism must therefore be based on a sound knowledge of its anatomy. Experiments can then be carried out to establish how particular parts perform their functions. Although there have been many important physiological investigations on human volunteers, the need for precise control over the experimental conditions has meant that much of our present physiological knowledge has been derived from studies on other animals such as frogs, rabbits, cats, and dogs. When it is clear that a specific physiological process has a common basis in a wide variety of animal species, it is reasonable to assume that the same principles will apply to humans. The knowledge gained from this approach has given us a great insight into human physiology and endowed us with a solid foundation for the effective treatment of many diseases. The building blocks of the body are the cells, which are grouped together to form tissues. The principal types of tissue are epithelial, connective, nervous, and muscular, each with its own characteristics. Many connective tissues have relatively few cells but have an extensive extracellular matrix. In contrast, smooth muscle consists of densely packed layers of muscle cells linked together via specific cell junctions. Organs such as the brain, the heart, the lungs, the intestines, and the liver are formed by the aggregation of different kinds of tissues. The organs are themselves parts of distinct physiological systems. The heart and blood vessels form the cardiovascular system; the lungs, trachea, and bronchi together with the chest wall and diaphragm form the respiratory system; the skeleton and skeletal muscles form the musculoskeletal system; the brain, spinal cord, autonomic nerves and ganglia, and peripheral somatic nerves form the nervous system, and so on. Cells differ widely in form and function but they all have certain 生理学简介 介绍 生理学是研究生物体功能的科学。它研究生物体如何进行各种活动，如何饮食，如何运动，如何适应不断改变的环境，如何繁殖后代。这门学科包罗万象，涵盖了生物体整个生命过程。生理学成功地解释了生物体如何进行日常活动，基于的观点是生物体好比是结构复杂而灵巧的机器，其操作受物理和化学规律控制。 尽管从生物学整个范畴看，生物体某些活动过程是相似的——如基因编码的复制——但许多过程还是某些生物体群组特有的。鉴于此有必要将这门学科分成不同部分研究，如细菌生理学、植物生理学和动物生理学。 要研究一种动物如何活动，首先需要了解它的构成。要充分了解一个生物体的生理学活动就必须掌握全面的解剖学知识。一个生物体的各部分起着什么作用可通过实验观察得知。尽管我们对志愿者进行了许多重要的生理调查，但是实验条件需要精确控制，所以我们当前大多生理知识还是源于对其它动物如青蛙，兔子，猫和狗等的研究。当我们明确大多数动物物种的特定生理过程存在共同之处时，相同的生理原理适用于人类也是合理的。通过这种方法，我们获得了大量的知识，从而让我们对人类生理学有了更深入的了解，为我们有效治疗许多疾病提供了一个坚实的基础。 机体的基本组成物质是细胞，细胞结合在一起形成组织。组织的基本类型有上皮组织，结缔组织，神经组织和肌组织，每类组织都有各自的特征。许多结缔组织中细胞量相对较少，但是有大量的细胞外基质。相比而言，光滑的肌组织由大量密密麻麻的肌细胞通过特定的细胞连接组成。各种器官如脑，心脏，肺，小肠和肝等由不同种类的组织聚集而成。这些器官是不同生理系统的组成部分。心脏和血管组成心血管系统；肺，器官，支气管，胸壁和膈肌组成呼吸系统；骨骼和骨骼肌组成骨骼肌系统；大脑，脊髓，自主神经和神经中枢以及

药学英语专业词汇word精品

药学名词（中-英）6-磷酸葡萄糖脱氢酶glucose-6-phosphate dehydrogenase Janbon综合症Janbon's syndrome PPB浓度parts per billion concentration pphm 浓度parts per hundred million concentration PPH浓度parts per hundred concentration ppm 浓度parts per million concentration 安全范围safety range 安全试验法innocuity test method 安全系统safety coefficient 安慰剂placebo 螯合剂chelating agent 靶细胞target cell 白蛋白微球制剂albumin microballoons 百分浓度percentage concentration 半合成抗生素semisynthetic antibiotics 半抗原haptene 半数致死剂量half lethal dose ; median lethal dose; LD50 半衰期half-life period; half life time 包衣片coated tablet 薄膜衣film-coating 饱和溶液saturated solution 贝克勒尔Becquerel 被动免疫passive immunity 被动转运passive transport 崩解度disintegration 崩解剂disintegrants 必需氨基酸essential aminoacid 必需脂肪酸essential fatty acid 变态反应allergy; allergic reaction 表面活性surface activity 表面张力surface tension 丙种射线gamma rays 补体complement 补体系统complement system 不良反应adverse reaction 不完全抗原incomplete antigen 搽剂liniments 长期毒性实验long term toxicity test 长效制剂prolonged action preparation 肠肝循环enterohepatic circulation 肠溶控释片enteric controlled release tablets 肠溶衣enteric coating 处方prescription;recipe 穿透促进剂penetration enhancers 磁性控释制剂magnetic controlled release dosage form 磁性药物制剂magnetic medicinal preparations 大分子macromolecule 单克隆抗体monoclonal antibody 胆碱酯酶cholinesterase 当量equivalent weight 当量定律equivalent law 当量浓度normality 当量溶液normal solution 等张溶液isotonic solution 低聚糖oligosaccharides 低密度脂蛋白low density lipoprotein 滴定titration 滴定曲线titration curve 滴丸剂 pill 递质transmitter 电解electrolyzation 电解质electrolyte 酊剂tincture 定向药物制剂directed pharmaceutical preparations 毒理学toxicology 毒性反应toxic response; toxic reaction 短期致癌实验short term carcinogenic test 对因治疗etiological treatment 对映体antipode 对症治疗symptomatic treatment 多功能酶multifunctional enzyme 多剂量给药multiple dose administration 多糖polyose 多肽polypeptide 儿茶酚胺catecholamine 二重感染superinfection 发酵fermentation 法定处方official formula

药学英语第五版原文翻译

IntroductiontoPhysiology Introduction Physiologyisthestudyofthefunctionsoflivingmatter.Itisconcernedwithhowanorganismperformsitsv ariedactivities:howitfeeds,howitmoves,howitadaptstochangingcircumstances,howitspawnsnewgenerati ons.Thesubjectisvastandembracesthewholeoflife.Thesuccessofphysiologyinexplaininghoworganismsp erformtheirdailytasksisbasedonthenotionthattheyareintricateandexquisitemachineswhoseoperationisgo vernedbythelawsofphysicsandchemistry. Althoughsomeprocessesaresimilaracrossthewholespectrumofbiology—thereplicationofthegenetic codefororexample—manyarespecifictoparticulargroupsoforganisms.Forthisreasonitisnecessarytodivid ethesubjectintovariouspartssuchasbacterialphysiology,plantphysiology,andanimalphysiology. Tostudyhowananimalworksitisfirstnecessarytoknowhowitisbuilt.Afullappreciationofthephysiolog yofanorganismmustthereforebebasedonasoundknowledgeofitsanatomy.Experimentscanthenbecarriedo uttoestablishhowparticularpartsperformtheirfunctions.Althoughtherehavebeenmanyimportantphysiolo gicalinvestigationsonhumanvolunteers,theneedforprecisecontrolovertheexperimentalconditionshasmea ntthatmuchofourpresentphysiologicalknowledgehasbeenderivedfromstudiesonotheranimalssuchasfrog s,rabbits,cats,anddogs.Whenitisclearthataspecificphysiologicalprocesshasacommonbasisinawidevariet yofanimalspecies,itisreasonabletoassumethatthesameprincipleswillapplytohumans.Theknowledgegain edfromthisapproachhasgivenusagreatinsightintohumanphysiologyandendoweduswithasolidfoundation fortheeffectivetreatmentofmanydiseases. Thebuildingblocksofthebodyarethecells,whicharegroupedtogethertoformtissues.Theprincipaltype softissueareepithelial,connective,nervous,andmuscular,eachwithitsowncharacteristics.Manyconnective tissueshaverelativelyfewcellsbuthaveanextensiveextracellularmatrix.Incontrast,smoothmuscleconsists https://www.wendangku.net/doc/3f15973364.html,anssuchasthebrain,theh eart,thelungs,theintestines,andtheliverareformedbytheaggregationofdifferentkindsoftissues.Theorgans arethemselvespartsofdistinctphysiologicalsystems.Theheartandbloodvesselsformthecardiovascularsyst em;thelungs,trachea,andbronchitogetherwiththechestwallanddiaphragmformtherespiratorysystem;thes keletonandskeletalmusclesformthemusculoskeletalsystem;thebrain,spinalcord,autonomicnervesandgan glia,andperipheralsomaticnervesformthenervoussystem,andsoon. Cellsdifferwidelyinformandfunctionbuttheyallhavecertaincommoncharacteristics.Firstly,theyareb oundedbyalimitingmembrane,theplasmamembrane.Secondly,theyhavetheabilitytobreakdownlargemol eculestosmalleronestoliberateenergyfortheiractivities.Thirdly,atsomepointintheirlifehistory,theyposses sanucleuswhichcontainsgeneticinformationintheformofdeoxyribonucleicacid(DNA). Livingcellscontinuallytransformmaterials.Theybreakdownglucoseandfatstoprovideenergyforother activitiessuchasmotilityandthesynthesisofproteinsforgrowthandrepair.Thesechemicalchangesarecollect ivelycalledmetabolism.Thebreakdownoflargemoleculestosmalleronesiscalledcatabolismandthesynthes isoflargemoleculesfromsmalleronesanabolism. Inthecourseofevolution,cellsbegantodifferentiatetoservedifferentfunctions.Somedevelopedtheabil itytocontract(musclecells),otherstoconductelectricalsignals(nervecells).Afurthergroupdevelopedtheabi litytosecretedifferentsubstancessuchashormonesorenzymes.Duringembryologicaldevelopment,thispro cessofdifferentiationisre-enactedasmanydifferenttypesofcellareformedfromthefertilizedegg. Mosttissuescontainamixtureofcelltypes.Forexample,bloodconsistsofredcells,whitecells,andplatele ts.Redcellstransportoxygenaroundthebody.Thewhitecellsplayanimportantroleindefenseagainstinfection 生理学简介 介绍 生理学是研究生物体功能的科学。它研究生物体如何进行各种活动，如何饮食，如何运动，如何适应不断改变的环境，如何繁殖后代。这门学科包罗万象，涵盖了生物体整个生命过程。生理学成功地

药学英语专业词汇

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