Mary D. Boudreau

Photodecomposition of vitamin A and photobiological implications for the skin.

Vitamin A (retinol), an essential human nutrient, plays an important role in cellular differentiation, regulation of epidermal cell growth and normal cell maintenance. In addition to these physiological roles, vitamin A has a rich photochemistry. Photoisomerization of vitamin A, involved in signal transduction for vision, has been extensively investigated. The biological effects of light‐induced degradation of vitamin A and formation of reactive species are less understood and may be important for light‐exposed tissues, such as the skin. Photochemical studies have demonstrated that excitation of retinol or its esters with UV light generates a number of reactive species including singlet oxygen and superoxide radical anion. These reactive oxygen species have been shown to damage a number of cellular targets, including lipids and DNA. Consistent with the potential for damaging DNA, retinyl palmitate has been shown to be photomutagenic in an in vitro test system. The results of mechanistic studies were consistent with mutagenesis through oxidative damage. Vitamin A in the skin resides in a complex environment that in many ways is very different from the chemical environment in solution and in in vitro test systems. Relevant clinical studies or studies in animal models are therefore needed to establish whether the pro‐oxidant activity of photoexcited vitamin A is observed in vivo, and to assess the related risks.

Clear evidence of carcinogenic activity by a whole-leaf extract of Aloe barbadensis miller (aloe vera) in F344/N rats.

Aloe barbadensis Miller (Aloe vera) is an herbal remedy promoted to treat a variety of illnesses; however, only limited data are available on the safety of this dietary supplement. Drinking water exposure of F344/N rats and B6C3F1 mice to an Aloe vera whole-leaf extract (1, 2, and 3%) for 13 weeks resulted in goblet cell hyperplasia of the large intestine in both species. Based upon this observation, 2-year drinking water studies were conducted to assess the carcinogenic potential of an Aloe vera whole-leaf extract when administered to F344/N rats (48 per sex per group) at 0.5, 1, and 1.5%, and B6C3F1 mice (48 per sex per group) at 1, 2, and 3%. Compared with controls, survival was decreased in the 1.5% dose group of female rats. Treatment-related neoplasms and nonneoplastic lesions in both species were confined primarily to the large intestine. Incidences of adenomas and/or carcinomas of the ileo-cecal and cecal-colic junction, cecum, and ascending and transverse colon were significantly higher than controls in male and female rats in the 1 and 1.5% dose groups. There were no neoplasms of the large intestine in mice or in the 0 or 0.5% dose groups of rats. Increased incidences of mucosa hyperplasia of the large intestine were observed in F344/N rats, and increased incidences of goblet cell hyperplasia of the large intestine occurred in B6C3F1 mice. These results indicate that Aloe vera whole-leaf extract is an intestinal irritant in F344/N rats and B6C3F1 mice and a carcinogen of the large intestine in F344/N rats.

Differential Effects of Silver Nanoparticles and Silver Ions on Tissue Accumulation, Distribution, and Toxicity in the Sprague Dawley Rat Following Daily Oral Gavage Administration for 13 Weeks

There are concerns within the regulatory and research communities regarding the health impact associated with consumer exposure to silver nanoparticles (AgNPs). This study evaluated particulate and ionic forms of silver and particle size for differences in silver accumulation, distribution, morphology, and toxicity when administered daily by oral gavage to Sprague Dawley rats for 13 weeks. Test materials and dose formulations were characterized by transmission electron microscopy (TEM), dynamic light scattering, and inductively coupled mass spectrometry (ICP-MS). Seven-week-old rats (10 rats per sex per group) were randomly assigned to treatments: AgNP (10, 75, and 110 nm) at 9, 18, and 36 mg/kg body weight (bw); silver acetate (AgOAc) at 100, 200, and 400 mg/kg bw; and controls (2 mM sodium citrate (CIT) or water). At termination, complete necropsies were conducted, histopathology, hematology, serum chemistry, micronuclei, and reproductive system analyses were performed, and silver accumulations and distributions were determined. Rats exposed to AgNP did not show significant changes in body weights or intakes of feed and water relative to controls, and blood, reproductive system, and genetic tests were similar to controls. Differences in the distributional pattern and morphology of silver deposits were observed by TEM: AgNP appeared predominantly within cells, while AgOAc had an affinity for extracellular membranes. Significant dose-dependent and AgNP size-dependent accumulations were detected in tissues by ICP-MS. In addition, sex differences in silver accumulations were noted for a number of tissues and organs, with accumulations being significantly higher in female rats, especially in the kidney, liver, jejunum, and colon.

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays

Abstract The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

Levels of retinyl palmitate and retinol in the stratum corneum, epidermis, and dermis of female SKH-1 mice topically treated with retinyl palmitate.

Retinyl esters are the storage form of vitamin A in skin, and retinyl palmitate (RP) accounts for the majority of the retinyl esters endogenously formed in skin. RP is also obtained exogenously through the topical application of cosmetic and skin care products that contain RP. There is limited information on the penetration and distribution of RP and vitamin Awithin the stratified layers of the skin. The purpose of these studies was to determine the time course for accumulation and disappearance of RP and retinol in the stratified layers of skin from female SKH-1 mice that received single or repeated topical applications of creams containing 0.5 or 2% of RP. We developed an HPLC method with detection limits of 5.94 and 1.62 ng, to simultaneously quantify the amount of RP and retinol, respectively, in skin samples. Our results showed that RP rapidly diffuses into the stratum corneum and epidermal skin layers within 24 h following the application of RP-containing creams. Of the three skin layers, the highest level of RP and retinol per weight unit (ng/mg) at all time points was found in the epidermis. Levels of RP and retinol were lowest in the dermal layer and intermediate in the stratum corneum. The levels of RP and retinol in the separated skin layers and in the intact skin decreased with time, but levels of RP remained higher than control values for a period of up to 18 days. Our results indicate that the application of RP to mouse skin alters the normal physiological levels of RP and retinol in the skin.

Aloe vera Non-Decolorized Whole Leaf Extract-Induced Large Intestinal Tumors in F344 Rats Share Similar Molecular Pathways with Human Sporadic Colorectal Tumors

Aloe vera is one of the most commonly used botanicals for various prophylactic and therapeutic purposes. Recently, NTP/NCTR has demonstrated a dose-dependent increase in large intestinal tumors in F344 rats chronically exposed to Aloe barbadensis Miller (Aloe vera) non-decolorized whole leaf extract (AVNWLE) in drinking water. The morphological and molecular pathways of AVNWLE-induced large intestinal tumors in the F344 rats were compared to human colorectal cancer (hCRC) literature. Defined histological criteria were used to compare AVNWLE-induced large intestinal tumors with hCRC. The commonly mutated genes (Kras, Ctnnb1, and Tp53) and altered signaling pathways (MAPK, WNT, and TGF-β) important in hCRC were evaluated within AVNWLE-induced large intestinal tumors. Histological evaluation of the large intestinal tumors indicated eight of twelve adenomas (Ads) and four of twelve carcinomas (Cas). Mutation analysis of eight Ads and four Cas identified point mutations in exons 1 and 2 of the Kras gene (two of eight Ads, two of four Cas), and in exon 2 of the Ctnnb1 gene (three of eight Ads, one of four Cas). No Tp53 (exons 5–8) mutations were found in Ads or Cas. Molecular pathways important in hCRC such as MAPK, WNT, and TGF-β signaling were also altered in AVNWLE-induced Ads and Cas. In conclusion, the AVNWLE-induced large intestinal tumors in F344 rats share several similarities with hCRC at the morphological and molecular levels.

Levels of retinyl palmitate and retinol in stratum corneum, epidermis and dermis of SKH-1 mice.

Vitamin A (retinol) regulates many biological functions, including epidermal cell growth. Retinyl palmitate (RP) is the major esterified form of retinol and the predominant component of retinoids in the skin; however, how endogenous levels of RP and retinol in the skin are affected by the age of the animal remains unknown. Furthermore, the levels of retinol and RP in the various skin layers- the stratum corneum, epidermis and dermis of skin- have not been reported. In this paper, we report the development of a convenient method for separation of the skin from SKH-1 female mice into the stratum corneum, epidermis, and dermis and the determination of the levels of RP and retinol in the three fractions by HPLC analysis. The total quantities of RP and retinol from the stratum corneum, epidermis, and dermis are comparable to those extracted from the same amount of intact skin from the same mouse. There was an age-related effect on the levels of RP and retinol in the skin and liver of female mice. An age-related effect was also observed in the stratum corneum, epidermis, and dermis. The levels of RP and retinol were highest in the epidermis of 20-week-old mice, and decreased when the age increased to 60- and 68-weeks. The total amount of RP at 20 weeks of age was found to be 1.52 ng/mg skin, and decreased about 4-fold at 60- and 68-weeks of age. A similar trend was found for the effects of age on the levels of retinol.

Suppression of Arylamine Toxicity in the Fischer-344 Rat Following Ingestion of a Complex Mixture

The toxic effects of a mixture of 2-aminoanthracene (2-AA), benzanthracene (BA), and dinitropyrene isomers (DNP), and the toxic effects of these compounds individually, were investigated in the Fischer-344 rat following dietary exposure via a powdered basal diet. Animals were sacrifi ced at 14-, 30-, and 80-day s of dietary exposure. Exposure to dietary 2-AA alone induced anorexia, cachexia, variable mortality, and altered serum chemistry profi les in the F-344 rat. Reduced lymphocyte counts were also shown in rats exposed to 2-AA. A temporal pattern of effect of 2-AA dietary exposure was observed in the progression of hepatic lesions in exposed animals. Dietary exposure to either DNP isomers or BA at a 10-fold higher concentration in the diet, relative to 2-AA, did not induce detectable toxic responses. However, exposure of rats to a mixture of 2-AA, BA, and DNP isomers (100 mg/kg, 1.0 g/kg, and 1.0 g/kg of diet, respectively) resulted in the attenuation of toxic effects when compared to exposure of F-344 rats to 2-AA alone. These results indicate that the toxic effects of 2-AA are suppressed by co-administration of DNP and BA and suggest that compound interactions need to be considered when predicting the toxic potential of specifi c environmental pollutants.

From the Cover: Aloin, a Component of the Aloe Vera Plant Leaf, Induces Pathological Changes and Modulates the Composition of Microbiota in the Large Intestines of F344/N Male Rats

In a previous study, the oral administration of an Aloe vera whole leaf extract induced dose-related mucosal and goblet cell hyperplasia in the rat colon after 13 weeks and colon cancer after 2 years. The primary goal of this study was to determine whether or not the administration of aloin, a component of the Aloe vera plant leaf, would replicate the pathophysiological effects that were observed in rats in the previous study with an Aloe vera whole leaf extract. Groups of 10 male F344/N rats were administered aloin at 0, 6.95, 13.9, 27.8, 55.7, 111, 223, and 446 mg/kg drinking water for 13 weeks. At the end of study, rat feces were collected, and the composition of fecal bacteria was investigated by next generation sequencing of the PCR-amplified V3/V4 region of the 16S rRNA gene. At necropsy, blood was collected by cardiac puncture and organs and sections of the large intestine were collected for histopathology. Aloin induced dose-related increased incidences and severities of mucosal and goblet cell hyperplasia that extended from the cecum to the rectum, with increased incidences and severities detected at aloin doses ≥55.7 mg/kg drinking water. Analysis of the 16S rRNA metagenomics sequencing data revealed marked shifts in the structure of the gut microbiota in aloin-treated rats at each taxonomic rank. This study highlights the similarities in effects observed for aloin and the Aloe vera whole leaf extract, and points to a potential mechanism of action to explain the observed pathological changes via modulation of the gut microbiota composition.

“Nondecolorized” Qualifier Is a Misnomer for the Aloe Vera Whole Leaf Extract Test Material

The Letter to the Editor of Toxicological Sciences from the International Aloe Science Council (IASC) states that our article, which appeared in the January issue of Toxicological Sciences, “omits a vital test-material qualifier from the title and throughout the article.” The concern of the IASC is that the public will confuse the aloe vera whole leaf extract tested in this study with the “IASC industry standard aloe vera leaf products.” The published article clearly and accurately describes the test material as a whole leaf extract obtained from the intact leaves of the Aloe barbadensis Miller (Aloe vera) plant. The aloe vera whole leaf extract test material was described as “yellow in color and retains the full complement of the β-linked high-molecular-weight polysaccharides of the inner aloe vera leaf gel and the β-linked C-glycoside anthrones of the outer aloe vera leaf latex.” As such, no qualifier was deemed necessary to describe this material; the material tested was indeed the aloe vera whole leaf extract and contained all of the aloe vera plant leaf components, namely aloe vera gel and aloe vera latex. No other ingredients were added to the material tested, and the contents of marker compounds (malic acid, aloin A, and aloe-emodin) in the aloe vera whole leaf extract test material were clearly noted in Table 2 of the publication. The IASC has stated that the qualifier “is important because decolorized aloe vera leaf products containing less than 10 ppm aloin are the industry standard.” In their Letter to the Editor, the IASC stated that the difference between “decolorized aloe vera whole leaf extracts” (the industry standard) and the aloe vera whole leaf extract (the material tested) is that some of the components of the aloe vera whole leaf extract, primarily the anthrones and anthraquinones, have been removed in “decolorized aloe vera whole leaf extracts.” We understand the need for the IASC to use the qualifier “decolorized” to represent accurately the materials contained in the “industry standard” aloe vera leaf products because the “industry standard” does not contain all of the components of the aloe vera whole leaf extract. However, the material tested for this publication was the aloe vera whole leaf extract and to use the term “nondecolorized” is a misnomer, much the same as using the terms “nondefatted” milk for whole milk or “nondecaffeinated” coffee for regular coffee. In their Letter to the Editor, the IASC erroneously stated that the “nondecolorized” qualifier has been included in all previous publications issued by the NTP on this research. This is not correct. The IASC is correct to point out that the title to the NTP Technical Report (TR) 577 was changed as the result of public comments; however, two routes of exposure were examined in studies of aloe vera that were conducted at the National Center for Toxicological Research (NCTR): dermal and oral. The aloe vera leaf extracts used in the dermal photococarcinogenesis study included aloe vera gel, aloe vera whole leaf, and decolorized aloe vera whole leaf. These were the terms used in the NCTR study reports (August 2007) and NTP TR 553 (September 2010). No public comments or objections to the terms used for the aloe vera plant extracts were raised by the IASC before, during, or after the public review meeting that was held by the NTP Board of Scientific Counselors’ Technical Reports Review Subcommittee in February 2008 for NTP TR 553. The aloe vera leaf extracts used for the oral studies included aloe vera gel, aloe vera whole leaf, and decolorized aloe vera whole leaf. These were the terms used in the NCTR study reports (August 2010) and in the initial drafts of NTP TR 577 (January 2011). Public comments made by the IASC prior to the public review meeting for NTP TR 577 prompted the NTP staff to change the term aloe vera whole leaf to nondecolorized aloe vera whole leaf in the title and abstract of the review draft of NTP TR 577 (February 2011). The public review meeting for NTP TR 577 was held on April 5, 2011. In our Toxicological Sciences publication we used the term aloe vera whole leaf, which for the reasons stated above we believe best describes the material tested.