Fat mass and obesity associated (FTO) gene influences skeletal muscle phenotypes in non-resistance trained males and elite rugby playing position.

BACKGROUND: FTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes. METHODS: In 1089 participants, comprising 530 elite rugby athletes and 559 non-athletes, DNA was collected and genotyped for the FTO rs9939609 variant using real-time PCR. In a subgroup of non-resistance trained individuals (NT; n = 120), we also assessed structural and functional skeletal muscle phenotypes using dual energy x-ray absorptiometry, ultrasound and isokinetic dynamometry. In a subgroup of rugby athletes (n = 77), we assessed muscle power during a countermovement jump. RESULTS: In NT, TT genotype and T allele carriers had greater total body (4.8% and 4.1%) and total appendicular lean mass (LM; 3.0% and 2.1%) compared to AA genotype, with greater arm LM (0.8%) in T allele carriers and leg LM (2.1%) for TT, compared to AA genotype. Furthermore, the T allele was more common (94%) in selected elite rugby union athletes (back three and centre players) who are most reliant on LM rather than total body mass for success, compared to other rugby athletes (82%; P = 0.01, OR = 3.34) and controls (84%; P = 0.03, OR = 2.88). Accordingly, these athletes had greater peak power relative to body mass than other rugby athletes (14%; P = 2 x 10-6). CONCLUSION: Collectively, these results suggest that the T allele is associated with increased LM and elite athletic success. This has implications for athletic populations, as well as conditions characterised by low LM such as sarcopenia and cachexia.

The MMP3 gene in musculoskeletal soft tissue injury risk profiling: A study in two independent sample groups.

Matrix metalloproteinase-3 (MMP3) is a mediator of matrix remodelling and a proposed susceptibility locus in the genetic profile of musculoskeletal soft tissue injuries. Therefore, this study aimed to validate the MMP3 gene as a risk marker for these injuries by conducting a case control genetic association study in two independent samples groups. Three previously investigated MMP3 variants (rs679620, rs591058 and rs650108) in addition to the functional promoter variant (rs3025058) were genotyped in 195 Australian control participants and 79 Australian individuals with chronic Achilles tendinopathy. Similarly, 234 South African individuals with acute anterior cruciate ligament ruptures and 232 matched control participants were also analysed. Based on high linkage with the previously associated MMP3 variant rs679620, rs3025058 was inferred and found to be associated with increased risk for Achilles tendinopathy within the South African group (P = 0.012; OR: 2.88; 95% CI: 1.4 to 6.1). Lastly, the 6A-G-C-G haplotype, constructed from the investigated variants, was significantly associated with reduced risk for Achilles tendinopathy (29% CON vs. 20% TEN, P = 0.037) in the Australian group. In conclusion, a signal surrounding MMP3 is apparent with respect to Achilles tendinopathy. However, whether the investigated variants are contributing to injury susceptibility or whether they are merely linked to the risk conferring variants mapping elsewhere within the MMP gene cluster on chromosome 11, still requires refining.

Association of ACTN3 R577X but not ACE I/D gene variants with elite rugby union player status and playing position.

We aimed to quantify the ACE I/D and ACTN3 R577X (rs1815739) genetic variants in elite rugby athletes (rugby union and league) and compare genotype frequencies to controls and between playing positions. The rugby athlete cohort consisted of 507 Caucasian men, including 431 rugby union athletes that for some analyses were divided into backs and forwards and into specific positional groups: front five, back row, half backs, centers, and back three. Controls were 710 Caucasian men and women. Real-time PCR of genomic DNA was used to determine genotypes using TaqMan probes and groups were compared using χ(2) and odds ratio (OR) statistics. Correction of P values for multiple comparisons was according to Benjamini-Hochberg. There was no difference in ACE I/D genotype between groups. ACTN3 XX genotype tended to be underrepresented in rugby union backs (15.7%) compared with forwards (24.8%, P = 0.06). Interestingly, the 69 back three players (wings and full backs) in rugby union included only six XX genotype individuals (8.7%), with the R allele more common in the back three (68.8%) than controls (58.0%; χ(2) = 6.672, P = 0.04; OR = 1.60) and forwards (47.5%; χ(2) = 11.768, P = 0.01; OR = 2.00). Association of ACTN3 R577X with playing position in elite rugby union athletes suggests inherited fatigue resistance is more prevalent in forwards, while inherited sprint ability is more prevalent in backs, especially wings and full backs. These results also demonstrate the advantage of focusing genetic studies on a large cohort within a single sport, especially when intrasport positional differences exist, instead of combining several sports with varied demands and athlete characteristics.

ELN and FBN2 gene variants as risk factors for two sports-related musculoskeletal injuries.

The proteins ELN and FBN2 are important in extracellular matrix function. The ELN rs2071307 and FBN2 rs331079 gene variants have been associated with soft tissue pathologies. We aimed to determine whether these variants were predisposing factors for both Achilles tendinopathy (AT) and anterior cruciate ligament (ACL) ruptures. For the AT study, 135 cases (TEN group) and 239 asymptomatic controls were recruited. For the ACL rupture study our cohort consisted of 141 cases (ACL group) and 219 controls. Samples were genotyped for both the ELN rs2071307 and FBN2 rs331079 variants using TaqMan assays. Analysis of variance and chi-squared tests were used to determine whether either variant was associated with AT or ACL rupture with significance set at p<0.05. The GG genotype of the FBN2 variant was significantly over-represented within the TEN group (p=0.035; OR=1.83; 95% CI 1.04-3.25) compared to the CON group. We also found that the frequency of the G allele was significantly different between the TEN (p=0.017; OR=1.90; 95% CI 1.11-3.27) and ACL groups (p=0.047; OR=1.76; 95% CI 1.00-3.10) compared to controls. The ELN rs207137 variant was not associated with either AT or ACL rupture. In conclusion, DNA sequence variation within the FBN2 gene is associated with both AT and ACL rupture.

The GDF5 gene and anterior cruciate ligament rupture.

A recent genetic association study has revealed that a variant (rs143383) within the 5'-untranslated region of the growth differentiation factor 5 gene (GDF5) associates with the risk of Achilles tendon pathology. The aim of this study was to determine whether this variant associates with the risk of ACL rupture. A cohort of 126 Caucasians with ACL rupture (ACL group), including 51 subjects who ruptured their ACL through a non-contact mechanism (NON sub-group), and 214 controls (CON group) were genotyped for the rs143383 variant. We report no significant GDF5 rs143383 genotype (P=0.396) or allele (P=0.810) frequency differences between the ACL (TT genotype, n=37, 29%; CT genotype, n=72, 57%; CC genotype, n=17, 14%) and CON (TT genotype, n=73, 34%; CT genotype, n=106, 50%; CC genotype, n=35, 16%) groups. There were also no significant differences between the NON sub-group and the CON group (allele; P=0.710, genotype; P=0.771). Furthermore, in gender specific analysis we found no association between rs143383 and ACL in either males (allele; P=0.988, genotype; P=0.407) or females (allele; P=0.643, genotype; P=0.885), respectively. Nor were there any gender specific associations between the NON sub-group and either genotype or allele. In conclusion, the rs143383 variant was not found to associate with the risk of ACL rupture.

Matrix metalloproteinase genes on chromosome 11q22 and the risk of anterior cruciate ligament (ACL) rupture.

As matrix metalloproteinases (MMPs) are critical to ligament homeostasis and integrity, the aim of this study was to investigate whether four functional polymorphisms within four MMP genes, which cluster on chromosome 11q22 associate with risk of ACL ruptures. Three hundred and forty-five [129 with ACL ruptures (ACL group) and 216 asymptomatic controls (CON group)] unrelated Caucasians were recruited for this case-control study. Fifty-four participants reported non-contact mechanisms of ACL rupture (NON subgroup). All participants were genotyped for the MMP10 C/T rs486055, MMP1 1G/2G rs1799750, MMP3 G/A rs679620 and MMP12 A/G rs2276109 variants. After adjusting for sex, age and weight, the AG and GG genotypes of the MMP12 rs2276109 variant were significantly (P=0.030) under-represented among the NON subgroup (14%), when compared with the CON group (26%). No other variants were significantly different between groups. Adjusted for the same confounders, the two four-variant haplotypes T-1G-A-A (CON 14%, ACL 9%, P=0.033) and C-2G-G-G (CON 14%, NON 5%, P=0.021) were significantly different between the CON and the ACL groups, and the CON group and the NON subgroup, respectively. This is the first report that indicates an association between the chromosomal region 11q22 and the risk of ACL rupture.

Variants within the MMP3 gene are associated with Achilles tendinopathy: possible interaction with the COL5A1 gene.

OBJECTIVES: Sequence variation within the COL5A1 and TNC genes are known to associate with Achilles tendinopathy. The primary aim of this case-control genetic association study was to investigate whether variants within the matrix metalloproteinase 3 (MMP3) gene also contributed to both Achilles tendinopathy and Achilles tendon rupture in a Caucasian population. A secondary aim was to establish whether variants within the MMP3 gene interacted with the COL5A1 rs12722 variant to raise risk of these pathologies. METHODS: 114 subjects with symptoms of Achilles tendon pathology and 98 healthy controls were genotyped for MMP3 variants rs679620, rs591058 and rs650108. RESULTS: As single markers, significant associations were found between the GG genotype of rs679620 (OR = 2.5, 95% CI 1.2 to 4.90, p = 0.010), the CC genotype of rs591058 (OR = 2.3, 95% CI 1.1 to 4.50, p = 0.023) and the AA genotype of rs650108 (OR = 4.9, 95% CI 1.0 to 24.1, p = 0.043) and risk of Achilles tendinopathy. The ATG haplotype (rs679620, rs591058, and rs650108) was under-represented in the tendinopathy group when compared to the control group (41% vs 53%, p = 0.038). Finally, the G allele of rs679620 and the T allele of COL5A1 rs12722 significantly interacted to raise risk of AT (p = 0.006). No associations were found between any of the MMP3 markers and Achilles tendon rupture. CONCLUSION: Variants within the MMP3 gene are associated with Achilles tendinopathy. Furthermore, the MMP3 gene variant rs679620 and the COL5A1 marker rs12722 interact to modify the risk of tendinopathy. These data further support a genetic contribution to a common sports related injury.

Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol.

Repeated oral administration of chemopreventive retinoids such as isotretinoin over extended periods of time is associated with intolerable systemic toxicity. Here isotretinoin was formulated as a powder aerosol, and its delivery to the lungs of rats was studied with the aim to explore the possibility of minimizing adverse effects associated with its oral administration. Rats received isotretinoin orally (0.5, 1 or 10 mg kg(-1)) or by inhalation (theoretical dose approximately 1 or approximately 10 mg kg(-1)) in a nose-only inhalation chamber. Isotretinoin was quantitated by high-pressure liquid chromatography in plasma and lung tissue. The ratios of mean area of concentration-vs-time curve (AUC) values in the lungs over mean AUCs in the plasma for isotretinoin following single or repeated aerosol exposure surpassed those determined for the oral route by factors of between two (single low-dose) and five (single high-dose). Similarly, the equivalent ratios for the maximal peak concentrations in lungs and plasma obtained after aerosol exposure consistently exceeded those seen after oral administration, suggesting that lungs were exposed to higher isotretinoin concentrations after aerosol inhalation than after oral administration of similar doses. Repeated high doses of isotretinoin by inhalation resulted in moderate loss of body weight, but microscopic investigation of ten tissues including lung and oesophagus did not detect any significant aerosol-induced damage. The results suggest that administration of isotretinoin via powder aerosol inhalation is probably superior to its application via the oral route in terms of achieving efficacious drug concentrations in the lungs.

Rat cytochromes P450 (CYP) specifically contribute to the reductive bioactivation of AQ4N, an alkylaminoanthraquinone-di-N-oxide anticancer prodrug.

1. The bioreductive activation of the alkylaminoanthraquinone di-N-oxide prodrug AQ4N has been characterized in rat hepatic tissue using HPLC. 2. AQ4N was shown to be metabolized to two products, namely AQM, the two electron reduced mono-N-oxide, and AQ4, the four electron reduced active cytotoxic agent. 3. Metabolism was shown to occur in microsomes with an apparent Km = 30.29 microM and Vmax = 1.05 nmol/mg/min. 4. Bioreduction was dependent on anaerobic conditions and the presence of the reduced cofactor NADPH. Ketoconazole (100 microM) and carbon monoxide both inhibited AQ4N metabolism inferring a role for cytochrome P450 (CYP). 5. Microsomes from phenobarbitone and isoniazid-pretreated animals significantly (p < 0.05) enhanced the formation of AQ4 from AQ4N indicating a role for CYP2B and 2E respectively. The involvement of both CYP2B and 2E was confirmed by the use of CYP-specific inhibitors. 6. In conclusion, the involvement of rat hepatic CYP in the reductive bioactivation of the novel antitumour prodrug AQ4N has been established in detail for the first time. These findings highlight an important interspecies difference between the metabolism of AQ4N in rat and man which was shown earlier to be mediated by CYP3A enzymes. The pharmacological significance of this is discussed.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation.

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug.

PURPOSE: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N. METHODS AND MATERIALS: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression. RESULTS: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (+/- SE) for total AQ4N turnover was 14.26 +/- 1.43 nmol/incubate (highest) to 3.65 +/- 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70,p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism. CONCLUSIONS: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.

Alteration of transcription factor mRNAs during the isolation and culture of rat hepatocytes suggests the activation of a proliferative mode underlies their de-differentiation.

The commonly observed loss of liver specific phenotype regularly described in rat hepatocyte culture is typified by the loss of total cytochrome P450 (CYP) content and the altered abundance of CYP mRNAs. The current work shows that these changes are preceded by the induction of the mRNA encoding the transcription factor c-jun during the hepatocyte isolation procedure. Then as the hepatocytes attach to the substratum the induced expression of c-jun subsides and two patterns of CYP mRNA loss are observed. The mRNAs encoding CYPs 2C11, 2C13, 2E1, 3A1, 3A2 and 4A1 continuously decline while CYP 1A2, 2A1/2 and 2B1/2 mRNAs are temporarily stabilised for 2 to 2.5 hours at a reduced level before declining further. The loss of CYP1A2 and 2B1/2 mRNAs parallels the loss of the mRNAs encoding the liver specific transcription factors C/EBP alpha and HNF-1. The early and rapid increase in c-jun mRNA followed by a decline in C/EBP alpha mRNA are characteristic of the changes in the expression of these transcription factor mRNAs following the stimulation of hepatocyte proliferation after partial hepatectomy. The finding that the rate of loss of total P450 following partial hepatectomy parallels that in rat hepatocyte culture suggests that the commonly employed hepatocyte isolation procedure "primes" the normally quiescent hepatocytes to enter the cell cycle and de-differentiate especially as both systems lose the major constitutively expressed CYP2C11 isozyme.