title: Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay.
abstract: OBJECTIVE
  The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured.
  
  
  METHODS
  Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA).
  
  
  RESULTS
  Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production.
  
  
  CONCLUSIONS
  Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results.
  
  
  CONCLUSIONS
  Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples.
  
  
  CONCLUSIONS
  Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.
authors.affiliation: Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA. tg3@sanger.ac.uk
authors.forename: Tannia
authors.initials: T
authors.lastname: Gracia
authors.forename: Paul D
authors.initials: PD
authors.lastname: Jones
authors.forename: Eric B
authors.initials: EB
authors.lastname: Higley
authors.forename: Klara
authors.initials: K
authors.lastname: Hilscherova
authors.forename: John L
authors.initials: JL
authors.lastname: Newsted
authors.forename: Margaret B
authors.initials: MB
authors.lastname: Murphy
authors.forename: Alice K Y
authors.initials: AK
authors.lastname: Chan
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.forename: Markus
authors.initials: M
authors.lastname: Hecker
authors.forename: Paul K S
authors.initials: PK
authors.lastname: Lam
authors.forename: Rudolf S S
authors.initials: RS
authors.lastname: Wu
authors.forename: John P
authors.initials: JP
authors.lastname: Giesy
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002681:China; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000235:genetics / Q000378:metabolism; D005843:Geography; D006723:Hong Kong; D006801:Humans; D011374:Progesterone / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018570:Risk Assessment; D012722:Sewage / Q000032:analysis* / Q000737:chemistry; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013739:Testosterone / Q000235:genetics / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity*
chemical_list: D012333:RNA, Messenger; D012722:Sewage; D014874:Water Pollutants, Chemical; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D013250:Steroid Hydroxylases; D001141:Aromatase; D013254:Steroid 17-alpha-Hydroxylase