Dragon 1 Protocol Manuscript: Training, Accreditation, Implementation and Safety Evaluation of Portal and Hepatic Vein Embolization (PVE/HVE) to Accelerate Future Liver Remnant (FLR) Hypertrophy.

STUDY PURPOSE: The DRAGON 1 trial aims to assess training, implementation, safety and feasibility of combined portal- and hepatic-vein embolization (PVE/HVE) to accelerate future liver remnant (FLR) hypertrophy in patients with borderline resectable colorectal cancer liver metastases. METHODS: The DRAGON 1 trial is a worldwide multicenter prospective single arm trial. The primary endpoint is a composite of the safety of PVE/HVE, 90-day mortality, and one year accrual monitoring of each participating center. Secondary endpoints include: feasibility of resection, the used PVE and HVE techniques, FLR-hypertrophy, liver function (subset of centers), overall survival, and disease-free survival. All complications after the PVE/HVE procedure are documented. Liver volumes will be measured at week 1 and if applicable at week 3 and 6 after PVE/HVE and follow-up visits will be held at 1, 3, 6, and 12 months after the resection. RESULTS: Not applicable. CONCLUSION: DRAGON 1 is a prospective trial to assess the safety and feasibility of PVE/HVE. Participating study centers will be trained, and procedures standardized using Work Instructions (WI) to prepare for the DRAGON 2 randomized controlled trial. Outcomes should reveal the accrual potential of centers, safety profile of combined PVE/HVE and the effect of FLR-hypertrophy induction by PVE/HVE in patients with CRLM and a small FLR. TRIAL REGISTRATION: Clinicaltrials.gov: NCT04272931 (February 17, 2020). Toestingonline.nl: NL71535.068.19 (September 20, 2019).

Clinical added value of MRI to CT in patients scheduled for local therapy of colorectal liver metastases (CAMINO): study protocol for an international multicentre prospective diagnostic accuracy study.

BACKGROUND: Abdominal computed tomography (CT) is the standard imaging method for patients with suspected colorectal liver metastases (CRLM) in the diagnostic workup for surgery or thermal ablation. Diffusion-weighted and gadoxetic-acid-enhanced magnetic resonance imaging (MRI) of the liver is increasingly used to improve the detection rate and characterization of liver lesions. MRI is superior in detection and characterization of CRLM as compared to CT. However, it is unknown how MRI actually impacts patient management. The primary aim of the CAMINO study is to evaluate whether MRI has sufficient clinical added value to be routinely added to CT in the staging of CRLM. The secondary objective is to identify subgroups who benefit the most from additional MRI. METHODS: In this international multicentre prospective incremental diagnostic accuracy study, 298 patients with primary or recurrent CRLM scheduled for curative liver resection or thermal ablation based on CT staging will be enrolled from 17 centres across the Netherlands, Belgium, Norway, and Italy. All study participants will undergo CT and diffusion-weighted and gadoxetic-acid enhanced MRI prior to local therapy. The local multidisciplinary team will provide two local therapy plans: first, based on CT-staging and second, based on both CT and MRI. The primary outcome measure is the proportion of clinically significant CRLM (CS-CRLM) detected by MRI not visible on CT. CS-CRLM are defined as liver lesions leading to a change in local therapeutical management. If MRI detects new CRLM in segments which would have been resected in the original operative plan, these are not considered CS-CRLM. It is hypothesized that MRI will lead to the detection of CS-CRLM in ≥10% of patients which is considered the minimal clinically important difference. Furthermore, a prediction model will be developed using multivariable logistic regression modelling to evaluate the predictive value of patient, tumor and procedural variables on finding CS-CRLM on MRI. DISCUSSION: The CAMINO study will clarify the clinical added value of MRI to CT in patients with CRLM scheduled for local therapy. This study will provide the evidence required for the implementation of additional MRI in the routine work-up of patients with primary and recurrent CRLM for local therapy. TRIAL REGISTRATION: The CAMINO study was registered in the Netherlands National Trial Register under number NL8039 on September 20th 2019.

Comparing practice and outcome of laparoscopic liver resection between high-volume expert centres and nationwide low-to-medium volume centres.

BACKGROUND: Based on excellent outcomes from high-volume centres, laparoscopic liver resection is increasingly being adopted into nationwide practice which typically includes low-medium volume centres. It is unknown how the use and outcome of laparoscopic liver resection compare between high-volume centres and low-medium volume centres. This study aimed to compare use and outcome of laparoscopic liver resection in three leading European high-volume centres and nationwide practice in the Netherlands. METHOD: An international, retrospective multicentre cohort study including data from three European high-volume centres (Oslo, Southampton and Milan) and all 20 centres in the Netherlands performing laparoscopic liver resection (low-medium volume practice) from January 2011 to December 2016. A high-volume centre is defined as a centre performing >50 laparoscopic liver resections per year. Patients were retrospectively stratified into low, moderate- and high-risk Southampton difficulty score groups. RESULTS: A total of 2425 patients were included (1540 high-volume; 885 low-medium volume). The median annual proportion of laparoscopic liver resection was 42.9 per cent in high-volume centres and 7.2 per cent in low-medium volume centres. Patients in the high-volume centres had a lower conversion rate (7.4 versus 13.1 per cent; P < 0.001) with less intraoperative incidents (9.3 versus 14.6 per cent; P = 0.002) as compared to low-medium volume centres. Whereas postoperative morbidity and mortality rates were similar in the two groups, a lower reintervention rate (5.1 versus 7.2 per cent; P = 0.034) and a shorter postoperative hospital stay (3 versus 5 days; P < 0.001) were observed in the high-volume centres as compared to the low-medium volume centres. In each Southampton difficulty score group, the conversion rate was lower and hospital stay shorter in high-volume centres. The rate of intraoperative incidents did not differ in the low-risk group, whilst in the moderate-risk and high-risk groups this rate was lower in high-volume centres (absolute difference 6.7 and 14.2 per cent; all P < 0.004). CONCLUSION: High-volume expert centres had a sixfold higher use of laparoscopic liver resection, less conversions, and shorter hospital stay, as compared to a nationwide low-medium volume practice. Stratification into Southampton difficulty score risk groups identified some differences but largely outcomes appeared better for high-volume centres in each risk group.

Multicentre analysis of the learning curve for laparoscopic liver resection of the posterosuperior segments.

BACKGROUND: Laparoscopic liver resection demands expertise and a long learning curve. Resection of the posterosuperior segments is challenging, and there are no data on the learning curve. The aim of this study was to evaluate the learning curve for laparoscopic resection of the posterosuperior segments. METHODS: A cumulative sum (CUSUM) analysis of the difficulty score for resection was undertaken using patient data from four specialized centres. Risk-adjusted CUSUM analysis of duration of operation, blood loss and conversions was performed, adjusting for the difficulty score of the procedures. A receiver operating characteristic (ROC) curve was used to identify the completion of the learning curve. RESULTS: According to the CUSUM analysis of 464 patients, the learning curve showed an initial decrease in the difficulty score followed by an increase and, finally, stabilization. More patients with cirrhosis or previous surgery were operated in the latest phase of the learning curve. A smaller number of wedge resections and a larger number of anatomical resections were performed progressively. Dissection using a Cavitron ultrasonic surgical aspirator and the Pringle manoeuvre were used more frequently with time. Risk-adjusted CUSUM analysis showed a progressive decrease in operating time. Blood loss initially increased slightly, then stabilized and finally decreased over time. A similar trend was found for conversions. The learning curve was estimated to be 40 procedures for wedge and 65 for anatomical resections. CONCLUSION: The learning curve for laparoscopic liver resection of the posterosuperior segments consists of a stepwise process, during which accurate patient selection is key.

ANTECEDENTES: La resección hepática laparoscópica exige experiencia y una larga curva de aprendizaje. La resección de los segmentos posterosuperiores (PS) es un reto, y no hay datos acerca de la curva de aprendizaje (learning curve, LC). El objetivo de este estudio fue evaluar la LC de la resección laparoscópica de los segmentos PS. MÉTODOS: Se realizó un análisis CUSUM de la puntuación de dificultad (difficulty score, DS) de la resección en pacientes de 4 centros especializados. La técnica CUSUM se ajustó al riesgo (risk-adjusted CUSUM, RA-CUSUM) para el tiempo operatorio, la pérdida de sangre y las conversiones a cirugía abierta ajustando según la DS de los procedimientos. Se utilizó una curva ROC para identificar el momento en el que se consideró que la LC había sido completada. RESULTADOS: De acuerdo con el análisis CUSUM de los 464 pacientes incluidos, se observó una DS baja al inicio, que posteriormente se fue incrementando hasta llegar a una estabilización. En la última fase de la LC se operaron más pacientes con cirrosis o cirugía previa. De forma progresiva se fueron reduciendo el número de resecciones hepáticas en cuña y aumentando el de resecciones anatómicas. A lo largo del tiempo se introdujo el CUSA y la maniobra de Pringle con mayor frecuencia. El RA-CUSUM mostró una reducción progresiva del tiempo operatorio. La pérdida de sangre inicialmente aumentó ligeramente, luego se estabilizó y finalmente disminuyó con el tiempo. Una tendencia similar se observó para las conversiones. La LC se estimó en 40 casos para las resecciones en cuña y en 65 casos para las resecciones anatómicas. CONCLUSIÓN: La LC de la resección hepática laparoscópica de los segmentos PS es un proceso paso a paso durante el cual la selección del paciente es clave.

Quality of life from a randomized trial of laparoscopic or open liver resection for colorectal liver metastases.

BACKGROUND: Most treatments for cancer cause a decline in patients' health-related quality of life (HRQoL). Limiting this decline is a universal goal for healthcare providers. Using minimally invasive instead of open surgical techniques might be one way to achieve this. The aim of this study was to compare postoperative HRQoL after open and laparoscopic liver resection. METHODS: This was a predefined substudy of an RCT comparing open with laparoscopic liver resection. Patients with colorectal liver metastases were assigned randomly to open or laparoscopic parenchyma-sparing liver resection. HRQoL was assessed with the Short Form 36 questionnaire at baseline, and 1 and 4 months after surgery. RESULTS: A total of 280 patients were randomized, of whom 273 underwent surgery (129 laparoscopic, 144 open); 682 questionnaires (83.3 per cent) were available for analysis. One month after surgery, patients in the laparoscopic surgery group reported reduced scores in two HRQoL domains (physical functioning and role physical), whereas those in the open surgery group reported reduced scores in five domains (physical functioning, role physical, bodily pain, vitality and social functioning). Four months after surgery, HRQoL scores in the laparoscopic group had returned to preoperative levels, whereas patients in the open group reported reduced scores for two domains (role physical and general health). The between-group difference was statistically significant in favour of laparoscopy for four domains after 1 month (role physical, bodily pain, vitality and social functioning) and for one domain after 4 months (role physical). CONCLUSION: Patients assigned to laparoscopic liver surgery reported better postoperative HRQoL than those assigned to open liver surgery. For role limitations caused by physical health problems, patients in the laparoscopic group reported better scores up to 4 months after surgery. Registration number: NCT01516710 ( http://www.clinicaltrials.gov).

ANTECEDENTES: La mayoría de los tratamientos para el cáncer causan una disminución de la calidad de vida relacionada con la salud (health-related quality of life, HRQoL) de los pacientes. Limitar este declive es un objetivo universal para los proveedores de atención médica. El uso de técnicas quirúrgicas mínimamente invasivas en lugar de abiertas podría ser una forma de lograrlo. El objetivo de este estudio fue comparar la HRQoL postoperatoria después de la resección hepática abierta y laparoscópica. MÉTODOS: Se trata de un subestudio predefinido de un ensayo aleatorizado y controlado que comparó la resección hepática abierta con la laparoscópica. Los pacientes con metástasis hepáticas colorrectales se asignaron aleatoriamente al grupo de resección hepática con preservación de parénquima por vía abierta o por vía laparoscópica. La HRQoL se evaluó con el cuestionario abreviado SF-36 en el momento basal y al cabo de 1 y 4 meses después de la cirugía. RESULTADOS: Un total de 280 pacientes fueron aleatorizados, de los cuales 273 se sometieron a cirugía (129 = laparoscópica, 144 = abierta) y hubo 682 cuestionarios (83%) disponibles para el análisis. Un mes después de la cirugía, los pacientes del grupo de cirugía laparoscópica presentaron puntuaciones reducidas en dos items de HRQoL (función física y rol físico), mientras que los pacientes del grupo de cirugía abierta presentaron puntuaciones reducidas en cinco items (función física, rol físico, dolor corporal, vitalidad y función social). Cuatro meses después de la cirugía, el grupo de cirugía laparoscópica había vuelto a los niveles preoperatorios de la HRQoL, mientras que los pacientes del grupo de cirugía abierta presentaron puntuaciones reducidas para dos items (función física y salud general). La diferencia entre los grupos fue estadísticamente significativa a favor de la laparoscopia para cuatro items después de un mes de la cirugía (rol físico, dolor corporal, vitalidad y función social) y para un ítem (rol físico) después de cuatro meses. CONCLUSIÓN: Los pacientes asignados a cirugía hepática laparoscópica presentaron mejor HRQoL postoperatoria que los pacientes asignados a cirugía hepática abierta. Para las limitaciones de roles causadas por problemas físicos de salud, los pacientes de cirugía laparoscópica presentaron mejores puntuaciones a los cuatro meses tras la intervención quirúrgica.

Cost-effectiveness of liver transplantation in patients with colorectal metastases confined to the liver.

BACKGROUND: Patients with non-resectable colorectal metastases are currently treated with chemotherapy. However, liver transplantation can increase the 5-year survival rate from 9 to 56 per cent if the cancer is confined to the liver. The aim of this study was to estimate the cost-effectiveness of liver transplantation for colorectal liver metastases. METHODS: A Markov model with a lifetime perspective was developed to estimate the life-years, quality-adjusted life-years (QALYs), direct healthcare costs and cost-effectiveness for patients with non-resectable colorectal liver metastases who received liver transplantation or chemotherapy alone. RESULTS: In non-selected cohorts, liver transplantation increased patients' life expectancy by 3·12 life-years (2·47 QALYs), at an additional cost of €209 143, giving an incremental cost-effectiveness ratio (ICER) of €67 140 per life-year (€84 667 per QALY) gained. In selected cohorts (selection based on tumour diameter, time since primary cancer, carcinoembryonic antigen levels and response to chemotherapy), the effect of liver transplantation increased to 4·23 life-years (3·41 QALYs), at a higher additional cost (€230 282), and the ICER decreased to €54 467 per life-year (€67 509 per QALY) gained. Given a willingness to pay of €70 500, the likelihood of transplantation being cost-effective was 0·66 and 0·94 (0·23 and 0·67 QALYs) for non-selected and selected cohorts respectively. CONCLUSION: Liver transplantation was cost-effective but only for highly selected patients. This might be possible in countries with good access to grafts and low waiting list mortality.

Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug.

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25-fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46-47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time-dependent inhibition had no NPE and reasonable PPE rates (15-18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25-fold change in midazolam AUC appears appropriate.

Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase 1.

N-acetyltransferase 1 (NAT1) catalyses the activation and/or deactivation of aromatic and heterocyclic amine carcinogens. A genetic polymorphism in NAT1 is associated with an increased risk of various cancers and drug toxicities, but epidemiological investigations are severely compromised by a poor understanding of the relationship between NAT1 genotype and phenotype. Human reference NAT1*4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Large reductions in N- and O-acetyltransferase catalytic activities were observed for recombinant NAT1 allozymes encoded by NAT1*14B, NAT1*15, NAT1*17, NAT1*19 and NAT1*22. Each of these alleles exhibited NAT1 protein expression levels below the limit of detection as measured by Western blot. No differences between high and low activity NAT1 alleles were observed in relative mRNA expression or relative transformation efficiency. The recombinant NAT1 17 and NAT1 22 allozymes showed reduced intrinsic stability when compared with NAT1 4. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) N-acetylation was not catalysed by any of the NAT1 allozymes. Large differences in the metabolic activation via O-acetylation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) were noted for NAT1 allelic variants. The results of these studies suggest an important role for the NAT1 genetic polymorphism in metabolism of aromatic and heterocyclic amine carcinogens. Furthermore, these results suggest that low NAT1 phenotype results from NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein.

Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms.

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.

DNA adduct levels and absence of tumors in female rapid and slow acetylator congenic hamsters administered the rat mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine.

N-acetyltransferases (EC 2.3.1.5) catalyze O-acetylation of heterocyclic amine carcinogens to DNA-reactive electrophiles that bind and mutate DNA. An acetylation polymorphism exists in humans and Syrian hamsters regulated by N-acetyltransferase-2 (NAT2) genotype. Some human epidemiological studies suggest a role for NAT2 phenotype in predisposition to cancers related to heterocyclic amine exposures, including breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen prevalent in the human environment and induces a high incidence of mammary tumors in female rats. PhIP-induced carcinogenesis was examined in female rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus. In both rapid and slow acetylators, PhIP-DNA adduct levels were highest in pancreas, lower in heart, small intestine, and colon, and lowest in mammary gland and liver. Metabolic activation of N-hydroxy-PhIP by O-acetyltransferase was highest in mammary epithelial cells, lower in liver and colon, and lowest in pancreas. Metabolic activation of N-hydroxy-PhIP by O-sulfotransferase was low in liver and colon and below the limit of detection in mammary epithelial cells and pancreas. Unlike the rat, PhIP did not induce breast or any other tumors in female rapid and slow acetylator congenic hamsters administered high-dose PhIP (10 doses of 75 mg/kg) and a high-fat diet.

Oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) yields PhIP-DNA adducts but not tumors in male Syrian hamsters congenic at the N-acetyltransferase 2 (NAT2) locus.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen present in well-done meat. PhIP must undergo host-mediated bioactivation to exert its mutagenic and carcinogenic effects. Following N-hydroxylation, N-acetyltransferases catalyze the O-acetylation (activation) of N-hydroxy-PhIP to an electrophile causing DNA damage. A well-defined genetic polymorphism in N-acetyltransferase 2 (NAT2) activity exists in humans and the Syrian hamster. Since some human epidemiological studies suggest an association between acetylator genotype and cancer susceptibility in individuals who consume well done meats, this study was designed to investigate the specific role of acetylator genotype in PhIP-induced tumors using a Syrian hamster model congenic at the NAT2 locus. Following oral administration of PhIP to male rapid and slow acetylator Syrian hamsters, DNA adducts were identified in each tissue examined with levels in the relative order: pancreas > heart and urinary bladder > prostate, small intestine and transverse colon > ascending colon, liver, cecum, descending colon, and rectum. However, no tumors were observed in male rapid and slow acetylator congenic hamsters administered 11 oral doses of PhIP (75 mg/kg) and maintained on a high fat diet for one year.

DNA adduct levels and intestinal lesions in congenic rapid and slow acetylator syrian hamsters admi food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).

Epidemiological studies indicate that rapid acetylators with a high intake of well-done red meat have an increased risk of colorectal cancer. Arylamine N-acetyltransferase enzymes (E.C. 2.3.1.5) activate carcinogenic heterocyclic amines found in the crust of fried meat via O-acetylation of their N-hydroxylamines to reactive intermediates that bind covalently to DNA and produce mutations. Syrian hamsters as well as humans express two N-acetyltransferase isozymes (NAT1 and NAT2) which differ in substrate specificity and genetic control. Nucleic acid substitutions in the NAT2 gene segregate individuals into rapid, intermediate and slow acetylator phenotypes. In the present paper, we examined the role of the polymorphic NAT2 acetylator genotype in carcinogenesis induced by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by comparing Syrian hamster lines congenic at the NAT2 locus. No differences were found between rapid and slow acetylator congenic hamsters in levels of intestinal PhIP-DNA adducts. In contrast to previous studies in rats, no carcinogen-related induction of the preneoplastic lesions aberrant crypt foci or tumors was found in the intestines of rapid and slow acetylator congenic Syrian hamsters administered PhIP or IQ.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine induces a higher number of aberrant crypt foci in Fischer 344 (rapid) than in Wistar Kyoto (slow) acetylator inbred rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine carcinogen in the human diet and is a colon carcinogen in the rat. N-Acetyltransferase-2 (NAT2) catalyzes the conversion of PhIP and other heterocyclic amines to a DNA-reactive form. NAT2 has a polymorphic distribution in humans and other mammals, including rats. The rapid NAT2 genotype has been shown to be associated with increased colorectal cancer risk in some, but not all, human epidemiological studies. This investigation was designed to study the role of acetylator genotype in PhIP-induced colon carcinogenesis using aberrant crypt foci (ACF) as an intermediate biomarker. Five-week-old male, rapid-acetylator Fischer 344 (F344) rats and slow-acetylator Wistar-Kyoto (WKY) rats were fed the semipurified AIN76A diet with 0.01% PhIP, 0.04% PhIP, or no PhIP (control) for 8 weeks. PhIP induced ACF in both rapid- and slow-acetylator rats; 0.04% PhIP induced more ACF than 0.01% PhIP. There was no difference in the number of ACF between rapid- and slow-acetylator rats that were fed 0.01% PhIP. However, 0.04% PhIP induced 2-fold higher ACF and a greater dose-dependent increase in PhIP-induced ACF in the rapid-acetylator F344 rats compared with the slow-acetylator WKY rats. The results support human epidemiological studies showing higher risk for colorectal cancer in rapid acetylators who frequently consume meat that is very well done.

Acetylator phenotype and genotype in patients infected with HIV: discordance between methods for phenotype determination and genotype.

The acetylator phenotype and genotype of AIDS patients, with and without an acute illness, was compared with that of healthy control subjects (30 per group). Two probe drugs, caffeine and dapsone, were used to determine the phenotype in the acutely ill cohort. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis served to distinguish between the 26 known NAT2 alleles and the 21 most common NAT1 alleles. The distribution (%) of slow:rapid acetylator phenotype seen among acutely ill AIDS patients differed with the probe substrate used: 70:30 with caffeine versus 53:47 with dapsone. Phenotype assignment differed considerably between the two methods and there were numerous discrepancies between phenotype and genotype. The NAT2 genotype distribution was 45:55 slow:rapid. Control subjects, phenotyped only with caffeine, were 67:33 slow:rapid versus 60:40 genotypically. Stable AIDS patients, phenotyped only with dapsone, were 55:45 slow:rapid versus 46:54 genotypically. Following resolution of their acute infections, 12 of the acutely ill subjects were rephenotyped with dapsone. Phenotype assignment remained unchanged in all cases. The distribution of NAT1 alleles was similar in all three groups. It is evident from the amount of discordance between caffeine phenotype and dapsone phenotype or genotype that caution should be exercised in the use of caffeine as a probe for NAT2 in acutely ill patients. It is also clear that meaningful study of the acetylation polymorphism requires both phenotypic and genotypic data.

Association between acetylator genotype and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adduct formation in colon and prostate of inbred Fischer 344 and Wistar Kyoto rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine (HCA) found in cooked meats, causes colon and prostate tumors in male rats. Polymorphic N-acetyltransferase metabolizes N-hydroxy-PhIP to a DNA-reactive form. Liver, colon, and prostate PhIP-DNA adduct levels were compared in male rapid-acetylator Fischer 344 (F344) and slow-acetylator Wistar-Kyoto (WKY) rats fed 0.01 or 0.04% PhIP. Liver PhIP-DNA adduct levels at both PhIP doses, and colon PhIP-DNA adduct levels at the 0.01% PhIP dose were unaffected by acetylator genotype. However, in rats fed 0.04% PhIP, colon PhIP-DNA adduct levels were higher in rapid acetylator F344 rats (P < 0.05). Similarly, prostate PhIP-DNA adduct levels were higher in rapid acetylator F344 rats at both PhIP doses (P < 0.05). The combination of the high-PhIP dose and rapid-acetylator genotype resulted in the highest level of PhIP-DNA adducts in rat colon and prostate.

Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms.

The focus of this review is the molecular genetics, including consensus NAT1 and NAT2 nomenclature, and cancer epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Two N-acetyltransferase isozymes, NAT1 and NAT2, are polymorphic and catalyze both N-acetylation (usually deactivation) and O-acetylation (usually activation) of aromatic and heterocyclic amine carcinogens. Epidemiological studies suggest that the NAT1 and NAT2 acetylation polymorphisms modify risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers. Associations between slow NAT2 acetylator genotypes and urinary bladder cancer and between rapid NAT2 acetylator genotypes and colorectal cancer are the most consistently reported. The individual risks associated with NAT1 and/or NAT2 acetylator genotypes are small, but they increase when considered in conjunction with other susceptibility genes and/or aromatic and heterocyclic amine carcinogen exposures. Because of the relatively high frequency of some NAT1 and NAT2 genotypes in the population, the attributable cancer risk may be high. The effect of NAT1 and NAT2 genotype on cancer risk varies with organ site, probably reflecting tissue-specific expression of NAT1 and NAT2. Ethnic differences exist in NAT1 and NAT2 genotype frequencies that may be a factor in cancer incidence. Large-scale molecular epidemiological studies that investigate the role of NAT1 and NAT2 genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 and NAT2 acetylation polymorphisms in cancer risk.

Epoxide hydrolases: biochemistry and molecular biology.

Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. In the case of xenobiotics and certain endogenous substances, epoxide intermediates have been implicated as ultimate mutagenic and carcinogenic initiators Adams et al. (Chem. Biol. Interact. 95 (1995) 57-77) Guengrich (Properties and Metabolic roles 4 (1982) 5-30) Sayer et al. (J. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2. 3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61-71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A(3) hydrolase, leukotriene A(4) hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.

Novel human N-acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype.

Three novel human NAT2 alleles (NAT2*5D, NAT2*6D, and NAT2*14G) were identified and characterized in a yeast expression system. The common rapid (NAT2*4) and slow (NAT2*5B) acetylator human NAT2 alleles were also characterized for comparison. The novel recombinant NAT2 allozymes catalyzed both N- and O-acetyltransferase activities at levels comparable with NAT2 5B and significantly below NAT2 4, suggesting that they confer slow acetylation phenotype. In order to investigate the molecular mechanism of slow acetylation in the novel NAT2 alleles, we assessed mRNA and protein expression levels and protein stability. No differences were observed in NAT2 mRNA expression among the novel alleles, NAT2*4 and NAT2*5B. However NAT2 5B and NAT2 5D, but not NAT2 6D and NAT2 14G protein expression were significantly lower than NAT2 4. In contrast, NAT2 6D was slightly (3.4-fold) and NAT2 14G was substantially (29-fold) less stable than NAT2 4. These results suggest that the 341T --> C (Ile(114) --> Thr) common to the NAT2*5 cluster is sufficient for reduction in NAT2 protein expression, but that mechanisms for slow acetylator phenotype differ for NAT2 alleles that do not contain 341T --> C, such as the NAT2*6 and NAT2*14 clusters. Different mechanisms for slow acetylator phenotype in humans are consistent with multiple slow acetylator phenotypes.

Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine identified in the human diet and in cigarette smoke that produces prostate tumors in the rat. PhIP is bioactivated by cytochrome P-450 enzymes to N-hydroxylated metabolites that undergo further activation by conjugation enzymes, including the N-acetyltransferases, NAT1 and NAT2. To investigate the role of prostate-specific expression of human N-acetyltransferase 2 (NAT2) on PhIP-induced prostate cancer, we constructed a transgenic mouse model that targeted expression of human NAT2 to the prostate. Following construction, prostate, liver, lung, colon, small intestine, urinary bladder, and kidney cytosols were tested for human NAT1- and NAT2-specific N-acetyltransferase activities. Human NAT2-specific N-acetyltransferase activities were 15-fold higher in prostate of transgenic mice versus control mice, but were equivalent between transgenic mice and control mice in all other tissues tested. Human NAT1-specific N-acetyltransferase activities did not differ between transgenic and control mice in any tissue tested. Prostate cytosols from transgenic and control mice did not differ in their capacity to catalyze the N-acetylation of 2-aminofluorene, the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-PhIP or the N,O-acetylation of N-hydroxy-2-acetylaminofluorene. Transgenic and control mice administered PhIP did not differ in PhIP-DNA adduct levels in the prostate. This study is the first to report transgenic expression of human NAT2 in the mouse. The results do not support a critical role for bioactivation of heterocyclic amine carcinogens by human N-acetyltransferase-2 in the prostate. However, the lack of an effect may relate to the level of overexpression achieved and the presence of endogenous mouse acetyltransferases and/or sulfotransferases.

Dietary selenium reduces the formation of aberrant crypts in rats administered 3,2'-dimethyl-4-aminobiphenyl.

Human epidemiologic studies suggest that low selenium status is associated with increased cancer risk and that selenium supplementation is associated with reduction in the incidence of several cancers, including colorectal cancer. Aromatic and heterocyclic amine carcinogens are thought to be important in the etiology of human colorectal cancer, but no information is available on the effects of selenium on aromatic amine-induced colon cancer. In order to investigate this effect, aberrant crypt foci (ACF), the putative preneoplastic lesions of colon cancer in humans and rodents, were used as a biomarker to test the hypothesis that selenium supplementation can reduce aromatic amine-induced colon carcinogenesis. Male weanling F344 inbred rats were fed a basal torula yeast selenium-deficient diet supplemented with 0, 0.1, or 2. 0 mg selenium/kg diet as selenite, selenate, or selenomethionine (SeMet). Animals were fed the diets for 4 weeks and then administered 1 sc injection/week for 2 weeks of 3, 2'-dimethyl-4-aminobiphenyl (DMABP; 100 mg/kg) or vehicle (peanut oil). At 12 weeks, the rats were euthanized and the colon and rectum were removed, opened longitudinally, and fixed in 70% ethanol. Glutathione peroxidase activities in erythrocytes and liver cytosol and selenium concentrations in the colon/rectum and kidney increased significantly (p < 0.05) and in a dose-dependent manner with each of the three selenium diets. No ACF were identified in vehicle-treated rats. In DMABP-treated rats, ACF frequencies decreased significantly (p < 0.05) in groups supplemented with 0.1 or 2.0 mg selenium/kg diet as selenite and selenate but not SeMet. There were no significant differences in ACF and aberrant crypts between rats fed 0.1 vs 2.0 mg selenium/kg diet. These results suggest that dietary selenium, depending on chemical form, can reduce aromatic amine-induced colon carcinogenesis.