Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Nitrogen balance in slow-growing Windsnyer pigs fed on incremental levels of amarula (Sclerocarya birrea subsp. caffra) nut cake.

Feeding-derived amarula cake to growing pigs can overcome a narrow range of ingredients challenges and improve productivity. The objective of the current study was to determine the response in nitrogen (N) balance in slow-growing pigs fed on incremental levels of amarula nut cake (ANC). Thirty clinically healthy male growing Windsnyer (30.7 kg ± 6.57) (mean ± standard deviation) were individually assigned to separate pens in a completely randomized design, with six pigs per dietary treatment. Iso-energetic experimental diets were formulated to contain 0, 50, 100, 150, and 200 g/kg dry matter (DM) of ANC using the summit and dilution technique. Pigs were given 10 days of dietary adaptation and a collection period of 5 consecutive days after 31 days of feeding. Nitrogen intake increased linearly with incremental levels of ANC (P < 0.01). As ANC inclusion increased, the nitrogen (N) absorption, apparent N digestibility, and N retention in pigs increased until it reached a maximum, then started to decrease (P < 0.05). Nitrogen utilization increased at the rate of 0.63 g for each 1 g increase in ANC (P < 0.01). There was a linear decrease (P < 0.01) in total nitrogen excretion through urine and faeces with ANC inclusion. Urinary pH levels decreased quadratically in response to graded levels of ANC (P < 0.01). The relationship between urinary pH and ANC inclusion was Y = 0.0115x2 - 0.3491x + 4.872 (P < 0.01). The nitrogen balance responses were due to ANC inclusion in diets that were balanced for limiting amino acids. It can be concluded that ANC reduces N excretion, potentially minimizing ammonia volatilization, which makes it an alternative protein source for slow-growing pigs.

Molecular detection of Babesia canis vogeli and Hepatozoon canis in dogs in the department of Magdalena (Colombia) / Detección molecular de Babesia canis vogeli y Hepatozoon canis en perros en el departamento del Magdalena (Colombia)

ABSTRACT The canine population in the cities of Ciénaga and Santa Marta has been estimated at 54,953 based on individual dogs with owners. Due to the role that dogs play in society, either as pets or as transmitters of zoonoses to humans, we conducted a study with 169 blood samples from dogs that visited two veterinary clinics in these locations between March and September of 2017. The objective of the study was to detect species of Babesia and Hepatozoon canis by amplifying the 18S gene using conventional polymerase chain reaction (PCRc). The presence of Babesia sp. and Hepatozoon canis was detected in 15 (8.87%) and 12 (7.10%) DNA samples, respectively. In addition, 7 (4.14%) cases of coinfection were recorded. The Babesia sp. sequences obtained corresponded to the B. canis vogeli subspecies. This both pathogens in the Colombian Caribbean region and cases of coinfection in Colombian dogs. Therefore, the national veterinary community is encouraged to consider the information presented here in their differential diagnoses associated with companion vector-borne diseases (CVBDs). This information will allow veterinary professionals to create control and prevention strategies to prevent the spread of these infections.

RESUMEN La población canina en las ciudades de Ciénaga y Santa Marta se ha estimado en 54.953 individuos con propietarios. Debido al rol que desempeñan los perros en la sociedad, ya sea como animales de compañía o como transmisores de zoonosis al humano, se realizó un estudio con 169 muestras sanguíneas de perros que visitaron dos clínicas veterinarias en estas localidades entre marzo y septiembre del año 2017. El objetivo del estudió consistió en detectar especies de Babesia y Hepatozoon canis amplificando el gen 18S mediante reacción en cadena de la polimerasa convencional (PCR-c). La presencia de Babesia sp. y Hepatozoon canis se detectó en 15 (8,87%) y 12 (7,10%) muestras de ADN, respectivamente. Además, se registraron 7 (4,14%) casos de coinfección. Las secuencias obtenidas de Babesia sp. correspondieron a la subespecie B. canis vogeli. Se presentan ambos patógenos para la región Caribe colombiana y casos de coinfección en perros de Colombia. Por lo tanto, se exhorta a la comunidad veterinaria nacional a considerar la información presentada en sus diagnósticos diferenciales asociados a las enfermedades transmitidas por vectores de compañía (CVBDs). Esta información permitirá a los profesionales veterinarios crear estrategias de control y prevención para mitigar la propagación de estas infecciones.

Dietary enzyme addition on the growth performance and carcass characteristics of pigs fed diets containing avocado oil cake silage.

Avocado oil cake (AOC) was mixed with dried grape pomace and sugarcane molasses and ensiled for 90 days. A total mixed ration containing 5% AOC silage was formulated and top dressed with Axtra® XB enzyme at 0, 2.5 and 5%. The experimental diets were fed to 24 (8 pigs/diet) Large White × Landrace (LW × LR) cross pigs (± 22-kg live weight). Growth performance data was recorded for 60 days, after which the pigs were adapted to chromic oxide mixed diet for 3 days, whereby faeces were collected for 5 days after to determine nutrient digestion. Following nutrient digestion, pigs were fasted for 12 h, weighed and slaughtered. Carcass samples were collected and analysed for meat quality. Dietary addition of enzyme increased (P < 0.05) dry matter intake and nutrient digestibility, but did not affected (P > 0.05) feed conversion ratio and average daily gain. Carcass characteristics were not affected (P > 0.05); however, small and large intestine weight and length were increased (P < 0.05) with enzyme inclusion in feed. Dietary treatments did not affect (P > 0.05) the colour and cooking quality of the meat. Enzyme addition was worth in the growth performance and nutrient digestion but did not affect the carcass characteristics and meat quality of pigs.

Development of a curated Hershberger database.

A systematic literature review was conducted to identify Hershberger bioassays for ∼3200 chemicals including those used to validate the OECD/US EPA guideline assay, US EPA's chemicals screened for endocrine activity, and the library of chemicals run in US EPA 's ToxCast in vitro assays. For 134 chemicals that met pre-defined criteria, experimental results were extracted into a database used to characterize uncertainty in results and evaluate the concordance of the Hershberger assay with other in vivo rodent studies that measure androgen-responsive endpoints. Of 25 chemicals tested in >1 Hershberger study, 28% had disagreements between studies (i.e. ≥1 positive and ≥1 negative study), and of the 65 chemicals tested in Hershberger studies and other in vivo studies with androgen-responsive endpoints, 43% indicated disagreements, though in some cases these may be explained by differences in study designs or physiology of the animal model. Ultimately, 49 chemicals were identified with reproducible androgen pathway responses confirmed in ≥2 in vivo rodent studies that could be considered reference chemicals useful for validating alternative methods.

Evaluation of androgen assay results using a curated Hershberger database.

A set of 39 reference chemicals with reproducible androgen pathway effects in vivo, identified in the companion manuscript [1], were used to interrogate the performance of the ToxCast/Tox 21 androgen receptor (AR) model based on 11 high throughput assays. Cytotoxicity data and specificity confirmation assays were used to distinguish assay loss-of-function from true antagonistic signaling suppression. Overall agreement was 66% (19/29), with ten additional inconclusive chemicals. Most discrepancies were explained using in vitro to in vivo extrapolation to estimate equivalent administered doses. The AR model had 100% positive predictive value for the in vivo response, i.e. there were no false positives, and chemicals with conclusive AR model results (agonist or antagonist) were consistently positive in vivo. Considering the lack of reproducibility of the in vivo Hershberger assay, the in vitro AR model may better predict specific AR interaction and can rapidly and cost-effectively screen thousands of chemicals without using animals.

Voluntary feed intake and growth performance of slow-growing pigs fed on increasing levels of ensiled potato hash meal.

The objective of the study was to determine voluntary feed intake and growth performance of Windsnyer pigs fed on increasing levels of potato hash silage meal. Thirty-six growing Windsnyer pigs (19 kg ± 5.59) (mean ± standard deviation (SD)) were individually and randomly assigned to six experimental diets containing 0, 80, 160, 240, 320 and 400 g/kg DM of potato hash silage. Diets containing the potato hash silage were formulated using diet dilution method from 0 g/kg and 400 g/kg. Six pigs were fed on each diet ad libitum for 6 weeks. Average daily feed intake (ADFI), average daily gain (ADG), gain to feed (G/F) ratio, scaled feed intake (SFI) and scaled average daily gain (SADG) were measured weekly. Increasing levels of potato hash silage caused a decrease (P < 0.05) in ADG, G/F ratio and SADG. The ADFI interacted significantly (P < 0.05) with the inclusion level of potato hash silage and week of feeding. Pigs fed on 240 g/kg potato hash silage had greater ADFI in the second, third and fourth week of feeding. There was a quadratic increase (P < 0.05) in ADFI. There was a linear decrease (P < 0.05) in ADG and G/F ratio and SADG as the potato hash silage level increased. Using piecewise regression, potato hash silage can be included up to 240 g/kg DM in Windsnyer pigs without undermining growth performance.

LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma.

BACKGROUND AND PURPOSE: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co-administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP). EXPERIMENTAL APPROACH: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper-reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05-1 mg·mL(-1) ) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure. KEY RESULTS: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper-reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co-administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL(-1) reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin. CONCLUSIONS AND IMPLICATIONS: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.

Transcriptional responses in the rat nasal epithelium following subchronic inhalation of naphthalene vapor.

Male and female Fischer 344 rats were exposed to naphthalene vapors at 0 (controls), 0.1, 1, 10, and 30ppm for 6h/d, 5 d/wk, over a 90-day period. Following exposure, the respiratory epithelium and olfactory epithelium from the nasal cavity were dissected separately, RNA was isolated, and gene expression microarray analysis was conducted. Only a few significant gene expression changes were observed in the olfactory or respiratory epithelium of either gender at the lowest concentration (0.1ppm). At the 1.0ppm concentration there was limited evidence of an oxidative stress response in the respiratory epithelium, but not in the olfactory epithelium. In contrast, a large number of significantly enriched cellular pathway responses were observed in both tissues at the two highest concentrations (10 and 30ppm, which correspond to tumorigenic concentrations in the NTP bioassay). The nature of these responses supports a mode of action involving oxidative stress, inflammation and proliferation. These results are consistent with a dose-dependent transition in the mode of action for naphthalene toxicity/carcinogenicity between 1.0 and 10ppm in the rat. In the female olfactory epithelium (the gender/site with the highest incidences of neuroblastomas in the NTP bioassay), the lowest concentration at which any signaling pathway was significantly affected, as characterized by the median pathway benchmark dose (BMD) or its 95% lower bound (BMDL) was 6.0 or 3.7ppm, respectively, while the lowest female olfactory BMD values for pathways related to glutathione homeostasis, inflammation, and proliferation were 16.1, 11.1, and 8.4ppm, respectively. In the male respiratory epithelium (the gender/site with the highest incidences of adenomas in the NTP bioassay), the lowest pathway BMD and BMDL were 0.4 and 0.3ppm, respectively, and the lowest male respiratory BMD values for pathways related to glutathione homeostasis, inflammation, and proliferation were 0.5, 0.7, and 0.9ppm, respectively. Using a published physiologically based pharmacokinetic (PBPK) model to estimate target tissue dose relevant to the proposed mode of action (total naphthalene metabolism per gram nasal tissue), the lowest transcriptional BMDLs from this analysis equate to human continuous naphthalene exposure at approximately 0.3ppm. It is unlikely that significant effects of naphthalene or its metabolites will occur at exposures below this concentration.

Concentration- and time-dependent genomic changes in the mouse urinary bladder following exposure to arsenate in drinking water for up to 12 weeks.

Inorganic arsenic (As(i)) is a known human bladder carcinogen. The objective of this study was to examine the concentration dependence of the genomic response to As(i) in the urinary bladders of mice. C57BL/6J mice were exposed for 1 or 12 weeks to arsenate in drinking water at concentrations of 0.5, 2, 10, and 50 mg As/l. Urinary bladders were analyzed using gene expression microarrays. A consistent reversal was observed in the direction of gene expression change: from predominantly decreased expression at 1 week to predominantly increased expression at 12 weeks. These results are consistent with evidence from in vitro studies of an acute adaptive response that is suppressed on longer exposure due to downregulation of Fos. Pathways with the highest enrichment in gene expression changes were associated with epithelial-to-mesenchymal transition, inflammation, and proliferation. Benchmark dose (BMD) analysis determined that the lowest median BMD values for pathways were above 5 mg As/l, despite the fact that pathway enrichment was observed at the 0.5 mg As/l exposure concentration. This disparity may result from the nonmonotonic nature of the concentration-responses for the expression changes of a number of genes, as evidenced by the much fewer gene expression changes at 2 mg As/l compared with lower or higher concentrations. Pathway categories with concentration-related gene expression changes included cellular morphogenesis, inflammation, apoptosis/survival, cell cycle control, and DNA damage response. The results of this study provide evidence of a concentration-dependent transition in the mode of action for the subchronic effects of As(i) in mouse bladder cells in the vicinity of 2 mg As(i)/l.

Functional analysis of multiple genomic signatures demonstrates that classification algorithms choose phenotype-related genes.

Gene expression signatures of toxicity and clinical response benefit both safety assessment and clinical practice; however, difficulties in connecting signature genes with the predicted end points have limited their application. The Microarray Quality Control Consortium II (MAQCII) project generated 262 signatures for ten clinical and three toxicological end points from six gene expression data sets, an unprecedented collection of diverse signatures that has permitted a wide-ranging analysis on the nature of such predictive models. A comprehensive analysis of the genes of these signatures and their nonredundant unions using ontology enrichment, biological network building and interactome connectivity analyses demonstrated the link between gene signatures and the biological basis of their predictive power. Different signatures for a given end point were more similar at the level of biological properties and transcriptional control than at the gene level. Signatures tended to be enriched in function and pathway in an end point and model-specific manner, and showed a topological bias for incoming interactions. Importantly, the level of biological similarity between different signatures for a given end point correlated positively with the accuracy of the signature predictions. These findings will aid the understanding, and application of predictive genomic signatures, and support their broader application in predictive medicine.

The DEAD-box protein p72 regulates ERalpha-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERalpha-positive breast cancer.

The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17) have been shown to act as transcriptional co-activators for a diverse range of transcription factors, including oestrogen receptor-alpha (ERalpha). Here, we show that, although both proteins interact with and co-activate ERalpha in reporter gene assays, small interfering RNA-mediated knockdown of p72, but not p68, results in a significant inhibition of oestrogen-dependent transcription of endogenous ERalpha-responsive genes and oestrogen-dependent growth of MCF-7 and ZR75-1 breast cancer cells. Furthermore, immunohistochemical staining of ERalpha-positive primary breast cancers for p68 and p72 indicate that p72 expression is associated with an increased period of relapse-free and overall survival (P=0.006 and 0.016, respectively), as well as being inversely associated with Her2 expression (P=0.008). Conversely, p68 shows no association with relapse-free period, or overall survival, but it is associated with an increased expression of Her2 (P=0.001), AIB-1 (P<0.001) and higher tumour grade (P=0.044). Our data thus highlight a crucial role for p72 in ERalpha co-activation and oestrogen-dependent cell growth and provide evidence in support of distinct but important roles for both p68 and p72 in regulating ERalpha activity in breast cancer.

Gene expression changes following acute hydrogen sulfide (H2S)-induced nasal respiratory epithelial injury.

Hydrogen sulfide (H2S) is a naturally occurring gas that is also associated with several industries. The potential for widespread human inhalation exposure to this toxic gas is a public health concern. The nasal epithelium is especially susceptible to H2S-induced pathology. Injury to and regeneration of the nasal respiratory mucosa occurred in animals with ongoing H2S exposure, suggesting that the regenerated respiratory epithelium under-goes an adaptive response and becomes resistant to further injury. To better understand this response, ten-week-old male Sprague-Dawley rats were exposed nose-only to either air or 200 ppm H2S for three hours per day for one day or five consecutive days. Nasal respiratory epithelial cells at the site of injury and regeneration were laser capture microdissected, and gene expression profiles were generated at three, six, and twenty-four hours after the initial three-hour exposure and at twenty-four hours after the fifth exposure using the Affymetrix Rat Genome 230 2.0 microarray. Gene ontology enrichment analysis showed that H2S exposure altered gene expression associated with a variety of biological processes, including cell cycle regulation, protein kinase regulation, and cytoskeletal organization and biogenesis. Surprisingly, our results did not show a significant change in cytochrome oxidase gene expression or bioenergetics.

Relative safety of traditional agricultural tractor power take-off (PTO) drivelines compared to fluid power--a review.

Nearly all tractor PTO arrangements used today consist of a rotating mechanical shaft with two or more universal joints and splined couplings. Although this method of power transfer has been the standard for decades, it continues to be a hazard to farm workers. Commonly, PTO accidents involve the snagging of clothes, resulting in the victim being rapidly and violently drawn into (and around) the rotating shaft. Entanglement injuries are both common and severe in the agricultural workforce, with poor shielding maintenance as a contributing factor. If PTO loads were driven with fluid power, this entanglement hazard would be eliminated. With high-pressure injection injury being the principal hazard, the fluid power alternative appears to pose a lower risk in terms of both frequency of occurrence and severity of injury.

Electrochemical sensor for detection of unmodified nucleic acids.

We have developed a nucleic acid (NA) sensor based on mediated electrochemical oxidation of guanine residues. In this method, oligonucleotide probes are bound to a tin-doped indium oxide (ITO) electrode through a self-assembled phosphonate monolayer. The end carboxyl moiety of the monolayer is activated with carbodiimide and reacted with the amine group of a C6 alkyl linker which has been added to the 5'-end of the oligonucleotide probe. Upon hybridization of the complementary target NA, the hybrid is detected using a redox-active mediator, tris(2,2'-bipyridyl) ruthenium(II). We speculate that the monolayer does not impede electron-transfer since it contains many defect sites when assembled on a polycrystalline ITO surface. These defect sites are accessible to the mediator, but not to NA or proteins. The electrocatalytic current was a linear function of the amount of guanine bound at the electrode surface, with a detection limit of 120 amoles of guanine cm(-2) at 0.28 cm(2) ITO electrodes.

Identification of toxicologically predictive gene sets using cDNA microarrays.

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

A clonal growth model: time-course simulations of liver foci growth following penta- or hexachlorobenzene treatment in a medium-term bioassay.

A combination of experimental and simulation approaches were used to analyze the clonal growth of preneoplastic, enzyme-altered foci during liver carcinogenesis in an initiation-promotion regimen. Male Fisher 344 rats, 8 weeks of age, were initiated with a single dose (200 mg/kg, i.p.) of diethylnitrosamine (DEN). Beginning 2 weeks later, animals were exposed to daily gavage consisting of 0.1 mmol/kg pentachlorobenzene (PECB) or hexachlorobenzene (HCB) in corn oil vehicle for 6 weeks. Partial hepatectomy was performed 3 weeks after initiation. Experimental data including liver weight, hepatocyte density (number of hepatocytes/unit volume), 5-bromo-2'-deoxyuridine-labeling index for analysis of cell division rate, and number and volume of glutathione-S-transferase pi-positive foci were collected 23, 26, 28, 47, or 56 days after initiation. Model parameters describing liver growth were obtained directly from the experimental data. The probability of mutation/division of normal cells and the growth rate of initiated cells were inferred by a comparison of model outcomes with the observed time courses of foci development. To describe the time-dependent increases in foci volume and the concomitant reduction of foci number observed in all treatment groups, the calibrated model for the DEN controls incorporated the hypothesis of two initiated cell populations (referred to as A and B cells) within the framework of the two-stage model. The B cells are initiated cells that have a selective growth advantage under conditions that inhibit the growth of A cells and normal hepatocytes. The parameter values defined in the DEN controls were used to evaluate experiments involving the administration of PECB or HCB. Both PECB and HCB caused a significant increase in foci volume compared with the DEN controls. HCB treatments resulted in increased proliferation of normal hepatocytes, which was not observed for PECB under the same treatment regimen. The best description of the data resulted from the model incorporating the hypothesis that PECB and HCB promoted the growth of foci via increased net growth rates of B cells. We present here a biologically based clonal growth simulation platform to describe the growth of preneoplastic foci under experimental manipulations of initiation-promotion studies. This simulation work is an example of quantitative approaches that could be useful for the analysis of other initiation-promotion studies.

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice.

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.