Nutritional effects by beta-carotene in lung in males and females of control mice versus BCMO knockout mice

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,150-monooxygenase 1 knockout (Bcmo1-/-) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1-/- mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1-/- mice. Testosterone levels were higher after BC supplementation only in Bcmo1-/- mice, which had, unlike wild-type (Bcmo1+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice. The experimental setup has been described previously for females and males (Van Helden et al., CMLS 2010 and Carcinogenesis, 2011, resp.). Twelve female (fe) and 12 male (ma) B6129SF1 (Bcmo1+/+) and 12 female and 12 male B6;129S-Bcmo1tm1dnp (Bcmo1-/-) mice were used for the dietary intervention, which was conducted in accordance with the German animal protection laws by the guidelines of the local veterinary authorities. During the breeding and weaning periods of the mice, mothers were maintained on KLIBA 3430 chow containing 14,000 IU vitamin A/kg diet (Provima Kliba AG, Kaiseraugst, Switzerland). Five-week-old female and male Bcmo1+/+ and Bcmo1-/- mice were caged in groups containing two to four siblings per group and were maintained under environmentally controlled conditions (temperature 24 C, 12 h/12 h light/dark cycle). Mice had ad libitum access to feed and water. Basic feed consisted of the pelletized diet D12450B (Research Diets Inc, USA) with a fat content of 10%. The diet was modified to contain 1,500 IU vitamin A/kg of diet, which is a vitamin A-sufficient diet, and the control diet (control) was supplemented with water-soluble vehicle beadlets (DSM Nutritional Products Ltd., Basel, Switzerland) containing DL-alpha-tocopherol and ascorbyl palmitate as stabilizers, as well as carriers such as gelatin, corn oil, sucrose and starch. The BC diet (BC) was supplemented with identical water-soluble beadlets containing BC (DSM Nutritional Products Ltd., Basel, Switzerland) to generate 150 mg BC/kg diet. Beadlets were added by the manufacturer before low temperature pelletting. Feed pellets were color marked and stored at 4 C in the dark. After 14 weeks of dietary intervention, mice were killed after isoflurane and ketamin anesthesia and blood and lung tissue isolated. Lungs were rinsed in phosphate-buffered saline (PBS) and snap frozen in liquid nitrogen, and subsequently stored at -80 C. Transcriptomic analyses were performed using Agilent whole genome 4x44K gene expression arrays according to the manufacturer's procedure.