Little is known about the occurrence and the fate of veterinary drugs in the environment. Therefore, a liquid chromatography/tandem mass spectrometry method was developed and employed to investigate in detail the distribution and persistence of the frequently used tetracyclines and tylosin in a field fertilized with liquid manure on April 2000 and April 2001; soil sampling was performed in May 2000, November 2000, and May 2001. We detected 4.0 mg/kg tetracycline and 0.1 mg/kg chlortetracycline in the liquid manure of April 2000, as well as comparable amounts in the liquid manure of April 2001. In the soil samples of May 2001, the highest average concentrations of 86.2 (0-10 cm), 198.7 (10-20 cm), and 171.7 microg/kg (20-30 cm) tetracycline and 4.6-7.3 micro/kg chlortetracycline (all three sublayers) were found. At soil depths between 30 and 90 cm, as well as in soil or groundwater, tetracyclines could not be detected. In addition, oxytetracycline and tylosin could not be detected in any sample investigated. We conclude that tetracyclines enter the environment in significant concentrations via repeated fertilizations with liquid manure, build up persistent residues, and accumulate in soil. Therefore, tetracyclines may have a potential risk and investigations on the environmental effects of these antibiotics are necessary.

Nicotine and cotinine concentrations were measured in placental tissue during the first trimester, in amniotic fluid during the second trimester and in placental tissue and fetal serum at birth. These values were compared to the corresponding serum concentrations of the smoking mothers. Nicotine concentrations in the placentas (range 3.3-28 ng/g), in amniotic fluid (range 1.5-23 ng/ml) and in fetal serum (range 0.5- 25 ng/ml) were all higher than the corresponding maternal serum values: amniotic fluid/maternal vein serum concentration ratio 1.54 ± (SD) 0.27 (n = 23; week 16-24 of gestation), umbilical vein serum/matemal vein serum ratio 1.12 ± 0.30 (n = 26; at birth); placental tissue/matemal vein serum ratio 2.58 ± 1.30 (n = 17; at birth); these ratios were between 1.2 and 5 during week 10 of gestation (n = 3). The ratios did not depend on the time between the last cigarette smoked and sampling. Significant correlations were found between nicotine concentrations in amniotic fluid and maternal serum (r = 0.88), between fetal and maternal serum levels (r = 0.88) and between placental and maternal serum levels (r = 0.52). Cotinine concentrations in placental tissue (range 10-131 ng/g), amniotic fluid (range 5-188 ng/ml) and fetal serum (range 15-233 ng/ml) were lower than or similar to corresponding maternal serum levels. Our results indicate that the human fetus is exposed to higher nicotine concentrations than the smoking mothers.

Administration of the antiepileptic drug valproic acid (VPA) during early pregnancy can result in a 1-2% incidence of spina bifida aperta, a closure defect of the posterior neural tube in the human. The predominant defect produced by VPA in the mouse is exencephaly, a closure defect of the anterior neural tube. Recent experiments demonstrate that an appropriate dosing regimen (consecutive doses of VPA on day 9 of gestation) can also result in a low incidence of spina bifida aperta, and a high incidence of spina bifida occulta in the mouse as a potential animal model. Relatively high doses and concentrations of VPA are needed in the mouse to produce neural tube defects, the human appears to be more sensitive in this regard. Maximal concentrations and not AUC (area under the concentration-time curve) values correlate with the incidence of neural tube defects in the mouse which could in part be explained by saturation of plasma protein binding, increased free drug available for placental transfer and the embryonic neuroepithelium acting as a "deep compartment". It is likely that the parent drug and not a metabolite is the proximate teratogen. Structure-activity relationships show a strict structural requirement for high teratogenic potency: the molecule must contain an alpha-hydrogen atom, a carboxyl function, branching on carbon atom 2 with two chains containing 3 carbon atoms each for maximum activity. If these two carbon chains are different, then enantiomers are present such as the R- and S-enantiomers of 2-n-propyl-4-pentenoic acid (4-en-VPA), 2-n-propyl-4-pentynoic acid (4-yn-VPA) and 2-ethylhexanoic acid. These enantiomers were synthesized and shown to be significantly different in regard to teratogenic potency. Pharmacokinetic studies indicate that both enantiomers of each compound reach the embryo to the same degree. Therefore, the intrinsic teratogenic activity of the enantiomers differ, suggesting a stereoselective interaction between the drugs and a chiral structure within the embryo, is involved in the mechanism of action. In sharp contrast to the teratogenic effect, the anticonvulsant activity and neurotoxicity of this compound class show broad structural specificity, opening the possibility for development of novel antiepileptic agents with low teratogenic potency such as 2-n-propyl-2-pentenoic acid (2-en-VPA). The molecular mechanism of the teratogenicity of VPA is quite unknown; of the several hypothesis suggested, the interaction of VPA with embryonic folate metabolism is discussed here.

Recently we showed that tetracyclines tend to persist and may accumulate in sandy soils after repeated fertilizations with liquid manure. We continued these field investigations from 2001 to 2003 and observed no further accumulation of tetracyclines in soil, but found that the average tetracycline concentration remained higher than 150 microg/kg soil. From 2000 to 2002, approximately 330 g tetracycline, 7 g chlortetracycline, 28 g sulfamethazine, and 57 g sulfadiazine per hectare were transferred via liquid manure to the topsoil (0-30 cm). Nevertheless, no leaching of tetracyclines into deeper soil segments or groundwater was observed. Furthermore, we developed new analytical methods for the detection of various sulfonamides in liquid manure, soil, and groundwater. Investigation of the same fields used in the tetracycline study showed that sulfamethazine occurred in concentrations approximately two orders of magnitude lower than that of tetracycline in the plow layer. Although there apparently were very low concentrations of sulfamethazine in soil, we detected it in groundwater sampled by suction probes at 1.4 m below soil surface in the spring of 2002. Further investigations confirmed these findings. To our knowledge, this is the first direct evidence of continuous leaching of a veterinary drug from soil into groundwater under field conditions. We conclude that tetracyclines and sulfonamides show distinctly different environmental behaviors. One explanation may be their different sorption coefficients in soil, indicating (in part) their different mobilities in this ecosystem.

Valproic acid (VPA) is a widely used antiepileptic agent that is undergoing clinical evaluation for anticancer therapy. We assessed the effects of VPA on angiogenesis in vitro and in vivo. In human umbilical vein endothelial cells, therapeutically relevant concentrations of VPA (0.25 to 1 mM) inhibited proliferation, migration, and tube formation. VPA 1 mM inhibited endothelial cell proliferation by 51 Ϯ 5%, migration by 86 Ϯ 11%, and tube formation by 82 Ϯ 3%. These changes were preceded by the hyperacetylation of histone H4, indicating the inhibition of histone deacetylase (HDAC), and a decreased expression of the endothelial nitric-oxide synthase (eNOS). The inhibition of endothelial cell tube formation by VPA was prevented by addition of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The anticonvulsive active VPA derivative 2-ethyl-4-methylpentanoic acid, which does not inhibit HDAC, did not affect endothelial cell proliferation, tube formation, or eNOS expression. VPA was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay and in a Matrigel plug assay in mice. Embryos from VPA-treated mice showed disturbed vessel formation. These results indicate that therapeutic plasma levels of VPA inhibit angiogenesis by a mechanism involving a decrease in eNOS expression preceded by HDAC inhibition.

Seborrhoea and acne are exclusively human diseases and sebaceous gland differentiation is species specific. Therefore, fundamental research on human sebaceous cell function and control requires human in vitro models. The human sebocyte culture model, introduced in 1989, has been used in several studies to elucidate sebaceous gland activity and its regulation at the cellular level. Cultured human sebocytes have been shown to preserve important sebocytic characteristics, although they undergo an incomplete terminal differentiation in vitro. In vitro synthesis of free fatty acids without bacterial involvement and marked interleukin 1α expression at the mRNA and protein levels with no further induction by lipopolysaccharides lead to the assumption that human sebocytes may initiate acne lesions by an intrinsic mechanism. Androgens affected sebocyte activity in vitro in a manner dependent on the localization of the sebaceous glands. In vitro stimulation of sebocyte proliferation by androgens could be completely abolished by spironolactone. Cultured sebocytes strongly expressed type 1 5α-reductase and metabolized testosterone to androstenedione, 5α-androstanedione, 5α-dihydrotestosterone, androsterone and 5α-androstanediol, whereas the levels of 5α-reductase activity were probably not feedback regulated. 4,7β-Dimethyl-4-aza-5αcholestan-3-one, a type 1 5α-reductase inhibitor, induced an early, marked down-regulation of 5α-reductase activity in human sebocytes in vitro, while hydrofinasteride, a type 2 inhibitor, required 10³-fold higher concentrations to induce similar effects. Stimulation of sebocyte proliferation by insulin, thyroid-stimulating hormone and hydrocortisone indicates that the hormonal control of the sebaceous gland could be a complex mechanism. Retinoids inhibited sebocyte proliferation in a dose-dependent manner and down-regulated lipid synthesis and sebocyte differentiation in vitro. Isotretinoin was the most potent compound. On the other hand, vitamin A was found essential for sebocyte activity and differentiation in vitro and could be partially substituted by synthetic retinoids. The inhibitory effect of isotretinoin on sebocyte proliferation was barely affected by the presence of vitamin A. The low persistent isotretinoin levels or, more likely, the considerably elevated tretinoin concentrations detected in human sebocytes after treatment with isotretinoin in vitro may be responsible for the inhibitory effect of this compound on sebocyte activity.

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Pig-house dust originates from feed, bedding, feces, and the animals themselves. If the animals receive drugs such as antibiotics, residues of these substances may occur in manure, in the air, or on surfaces of the respective animal house. In a retrospective study, we investigated dust samples collected during two decades from the same piggery for the occurrence of various antibiotics. In 90% of these samples, we detected up to five different antibiotics, including tylosin, various tetracyclines, sulfamethazine, and chloramphenicol, in total amounts up to 12.5 mg/kg dust. High dust exposure in animal confinement buildings is believed to be a respiratory health hazard because of the high content of microorganisms, endotoxins, and allergens. Further risks may arise from the inhalation of dust contaminated with a cocktail of antibiotics. Apart from that, our data provide first evidence for a new route of entry for veterinary drugs in the environment.