Drug analysis

关于药物的作用分析

Fact Sheet             Equilenin

Guideline
Equilenin is an important component in Premarin, fully synthesized in 1940 by Alfred L. Wilds., which was usually used in hormone replacement therapy. The molecular structure shows as the figure 1. The original source of steroid hormones like equilenin in driking water most likely comes from sewage treatment plants, concentrated animal feeding operations, agriculture operations and pharmaceuticals. As for its physicochemical characteristics, though equilenin itself is most hydrophobic, it can form sulfate salt and dissolve in water very well. One recent review of conjugated equine estrogens for their efficacy, safety and mechanism of action contain the information of 10 estrogens including equilenin. From their study equilenin has unique biological effects either individually or in combinations of two or three other estrogens, for the prevention of CVD and neuro-degenerative diseases like Parkinson’s disease and AD in both aging women and men. However,apart from its potent biological effects, the draw backs is also pominent. A  number of observational studies suggest that the use of either equilenin alone or in combination with pro-gestin (HRT) in healthy women with aging range from 50 to 59 is associated with a increased risk of getting heart attacks. 
 
Figure1. The molecular structure of Equilenin.
General description
Equilenin is an estrogenic steroid hormone which serves as an important component in commercial pill—Premarin used to treat women with a hysterectomy. Equilenin itself is most hydrophobic, however, it can form sulfate salt and dissolve in water very well.have well dissolve characteristics. The occurrence of pharmaceuticals, especially hormone in aquatic environment may cause a lot of problems to the health of both human and aquatic lives. 

Currently, the use of pharmacological doses of premarin (5–20 μg/day in oral contraceptives , 1000 μg /day in hormone replacement therapy) to achieve positive health outcome s is both widespread and recognized as safe (Swan, 2000). However, the long-term exposure to this kind of conjugated estrogens has a potential risk of endometrial cancer based on the researches since 1975 ( Ziel H. K. et al., 1975; ANNEGERS J F, et al., 1977). And it had been proved that long-term use of estrogen would increases the possibility of getting strokes, heart attacks, blood clots and breast cancer (Brunner R L. et al., 2005). Some researchers even suggested that the increase of environmental estrogens might have a close relationship with the decline in human sex ratio recently (WHO /IPCS, 2002). 

The influence of equilenin on wildlife is significant as well. Many aquatic organisms are significantly more sensitive to chemical contaminants than people, due to both level of exposure
to the chemical and differences in internal systems. Equilenin can cause the feminization of some species of male fish (Gimeno et al., 1998a, 1998b; Batty and Lim, 1999). In 2009, Tyler, C.R. has also reported that 4.2 ng/L of equilenin in the water can induce the vitellogenin in aquatic organism of oncorhynchus mykiss (Tyler C R. et al., 2009). 

Receptor-binding assays measure binding of agonists or antagonists to a specific cellular receptor, which assess endocrine-disrupting activity at the molecular level of biological organization. Equilenin can specifically binds to estrogen receptor at low concentration, which has important functions in regulating important pathways for fertility and reproduction (Christen V. et al., 2010). By using sheep uterus estrogen receptors, Leusch had determined the sewage effluent of Queensland still have estrogenic activity (Leusch et al., 2005a). The steroid hormone equilenin is considered as high risk of highly active compound.


Australian significance
Many of the drugs, especially hormone drugs are designed to maintain biological activity even at very low concentration, which can be even lower than the limit of routine analytical method. (Geisy et al., 2002). Since the existence of equilenin in drinking water might threaten to public health, detecting and dealing the contamination of equilenin is very essential. 

The source of steroid hormones like equilenin most likely comes from sewage treatment plants, concentrated animal feeding operations and agriculture operations (Kolodziej E P. et al., 2004) The rapidly expanding scales of synthetic hormone in pharmaceuticals contribute to the contamination of drinking water as well.

According to the data of Australian guidelines for water recycling of 2008, the maximum concentration of equilenin detected in secondary treated sewage was 280 ng/L, while the guideline value provided was only 30 ng/L (Fletcher T D. et al., 2008; Natural Resource Management Ministerial Council, Canberra, 2008) much lower than that contains in treated water. However, the recommended drinking water guideline for human pharmaceutical suggested that the maximum concentration for equilenin should not exceed 0.278 μg/L (Falconer IR et al., 2003).

Because equilenin can exert effect at very low concentration, optimizing the detection methods and treatment methods are urgent.

Treatment of drinking water
The most common treatment of recycled water among world includes secondary treatment, membrane filtration, reverse osmosis, granular activated carbon (GAC) adsorption, chlorination and ultraviolet (UV) light disinfection (Khan and Roser, 2007).At the easiest level, treatment of most wastewater is conducted by sedimentation through separation of solids from liquids. By gradually transforming dissolved material into solids, usually a biological floc, then go to a sort of of purity process. Ultraviolet light disinfection and reverse osmosis have high efficiency in dealing with steroid hormone contaminate, which can destroy more than 90% biological activity, while microfiltration or chlorination only have poor performance on the treatment of these kind of compounds (Ternes and Joss et al., 2006; Snyder et al 2007).

For steroid hormones, one of the most common degradation pathways in waters is photodegradation (Yu-Chen et al, 2005). The half-life of equilenin was 2-3 hours in river water and the degradation rate increases with the increase of pH up to 7.6 (Zhang, Y. et al., 2007). The study of reclaimed water of Queensland based on biomarker assays have shown that UV can get rid of most estrogenic activity of equilenin while the sand filter can only reduce part of it (Leusch et al., 2005).

Studies have shown that manganese dioxide (MnO2) can be used as an effective oxidative to remove most of estrogenic activity of equilenin (de Rudder. et al., 2004).

Microbial degradation of steroid hormones is another important pathway. Biodegradation of steroid hormones were prone to occur under aerobic instead of anaerobic conditions. Higher temperature can also accelerate the process of degration (Jurgens, M. D. et al., 2002).

Many studies focused on using microbacteria to deal with the water have done these years. Gram-positive bacteria like Bacillus, Nocardia, Rhodococcus, Mycobacterium, Noccardia and gram-negtive bacteria like Comamonas, Pseudomonas seem to be promising (Fujii, K. et al., 2002).

At the same time, preventive measures should apply, especially to control industrial and pharmaceutical discharges to sewerage systems.

Method of identification and detection
Equilenin can maintain estrogenic activity even at low concentration, as a result, it would be very difficult for routine analytical method to detect active equilenin in drinking water. However, LC/MS/MS is a very sensitive method and can detect samples which are at nanogram scales. 

The detectiopn process shows as following. The water samples should be dechlorinated first, add surrogate standard to the sample before applying solid phase extraction. The compounds then pass through the solid phase extraction disks containing C18 functional groups and are eluted from solid phase by a small amount of methanol. Load sample to Gemini 3 μm NX-C18 HPLC column and then use LC/MS/MS for analyzing the concentration of equilenin in drinking water.

This method can detect equilenin as low as 4 ng/L within 15 minutes, and excellent linearity values can be achieved (Sueki H. Leung. et al., 2012).

Health consideration

One recent study investgated the metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells. The method they use was identifing the metabolites of equilenin by GC/MS metabolite standards and deuterium-labeled analogues as internal standards. The results showed the two cell lines share the same pathways of equilenin metabolism. And the metabolism is extrahepatic, estrogen-responsive tissues. This study indicate the overwhelmingly effect of equilenin in cancer indcution. In this way, in order to avoid the bad influcence of equilenin, the warnning is that reducing the injection of such drug and make sure the water is purfied within the sate limitation.

The occurrence of steroid hormones, such as equilenin, in aquatic environment may cause a lot of problems to the health of both human and aquatic lives. Since equilenin can specifically binds to estrogen receptor, which plays an essential role in regulating important pathways for fertility and reproduction, as a consequence, detecting and dealing the contamination of equilenin is very essential, otherwise, the possibility of side effects may get higher. 


References
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